Apoe immunotherapy

ABSTRACT

The present invention provides antibodies that preferentially bind to an ApoE(1-272) fragment relative to ApoE(1-299). These antibodies serve to reduce the toxicity of this fragment and find use in treatment and prophylaxis of a variety of neurological diseases.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims priority to U.S. Provisional Patent ApplicationNo. 61/440,284, filed Feb. 7, 2011, the contents of which areincorporated by reference in the entirety.

REFERENCE TO A SEQUENCE LISTING

The Sequence Listing written in fileSequence_Listing_for_(—)057450-410708.txt is 41,971 bytes and wascreated on Feb. 7, 2012. The information contained in this file ishereby incorporated by reference.

BACKGROUND OF THE INVENTION

Apolipoprotein E (ApoE), which maps to 19q13.2, encodes a protein with arole in lipid transport and cholesterol processing. The protein has anN-terminal domain (residues 1-191) containing a low density lipoproteinreceptor binding sites, a C-terminal domain (residues 216-299)containing the major lipid binding sites (residues 240-272) and a hingeregion between the two domains.

The ApoE gene has three allelic variants: ApoE4, ApoE3, and ApoE2. ApoE3(also referred to herein as “E4,” E3,” and “E2,” respectively) is themost common form and ApoE2 is the least common. The frequency of theApoE4 version of the gene in different populations varies, but is alwaysless than 30% and frequently 8%-15%. ApoE4 is known to be a strong riskfactor for Alzheimer's disease (AD). The presence of one copy of theallele increases the likelihood of acquiring the disease about threefoldand increases the onset of disease an average of 5 years. Two copies ofthe same allele increases the risk of AD about 8-fold and decreases theage of onset by an average of 10 years. Analysis of autopsies fromindividuals who have died with AD and have an ApoE4 allele show thatAβ-related pathologies such as plaques and cerebrovascular amyloid isgenerally increased in those individuals carrying the allele. ApoE as alipoprotein interacts with lipid particles and hence is involved inlipid transport in plasma, particularly cholesterol. It has beenreported to have a role in the maintenance and repair of neurons. Theassociation between ApoE4 and Alzheimer's disease may be mediated bymultiple mechanisms both Aβ-dependent and independent. ApoE4 hasreported to be associated with increased production and deposition of Aβand itself to form toxic aggregates and proteolytic fragments (Zhong etal., J. Biol. Chem. 284, 6027-6031 (2009); Mahley et al., PNAS 103,5644-5651 (2006)).

The E2, E3 and E4 isoforms each has differential capacity to transportlipids and has differential effects on AD with E4 increasing and E2decreasing the overall risk of AD. The three alleles each differ fromone another at only two residues in humans, positions 112 and 158. ApoE2 has a Cys at both sites. E3 has a Cys at 112 and Arg at 158, and E4has an Arg at both sites. These changes result in overall structuraldifferences in all three isoforms that appear to account for the ADdisease-causing propensities.

SUMMARY OF THE CLAIMED INVENTION

The invention provides an antibody, e.g., a monoclonal antibody, thatpreferentially binds to ApoE(1-272) relative to binding to ApoE(1-299).Optionally, the antibody binds to an epitope including residue 272 ofApoE(1-272). Optionally, the antibody binds to an epitope including afree carboxyl group of position 272 of ApoE(1-272). Optionally, theantibody binds to the E4 isoform of ApoE(1-272). The monoclonal antibodycan be a humanized, chimeric, veneered or human antibody. The monoclonalantibody can be an Fab fragment, single chain Fv, or single domainantibody. The isotype can be human IgG isotype, for example, IgG1, IgG2,or IgG4. Optionally there is at least one mutation in the constantregion.

The invention further provides a monoclonal antibody having the threelight chain CDRs as defined by Kabat and three heavy chain CDRs asdefined by Kabat of monoclonal antibody 12C12, 15E10 or 15G8. 12C12,15E10 and 15G8 are mouse antibodies characterized by a light chainvariable region of SEQ ID NO:10 and a heavy chain variable region of SEQID NO:9.

The invention further provides a monoclonal antibody comprising threelight chain CDRs and three heavy chain CDRs, each light chain CDR havingat least 90% sequence identity to a corresponding CDR designated as SEQID NO:22 (CDRL1), SEQ ID NO:23 (CDR L2), and SEQ ID NO:24 (CDR L3), andeach heavy chain CDR having at least 90% sequence identity to acorresponding CDR designated as SEQ ID NO:19 (CDR H1), SEQ ID NO:20 (CDRH2), and SEQ ID NO:21(CDR H3).

The invention further provides a monoclonal antibody comprising threelight chain Kabat CDRs and three heavy chain Kabat CDRs, each CDRdiffers in sequence from a corresponding CDR from 15G8 by no more than 6replacements, deletions or insertions, wherein 15G8 is a mouse antibodycharacterized by a light chain variable region of SEQ ID NO:10 and aheavy chain variable region of SEQ ID NO:9. In some antibodies, each CDRdiffers in sequence by no more than 1 replacement, deletion orinsertion. In some antibodies, each light chain CDR has a 100% sequenceidentity to a corresponding CDR from 15G8 and no heavy chain CDR differsin sequence by more than 6 replacements, deletions or insertions. Insome antibodies, each of light chain CDR L1, L2, L3 and CDR H1 and CDRH3 has a 100% sequence identity to a corresponding CDR from 15G8, andCDR H2 differs in sequence by no more than 6 replacements, deletions orinsertions. In some antibodies, the mature heavy chain variable regioncomprises an amino acid sequence of SEQ ID NO:9 and the mature lightchain variable region comprises an amino acid sequence of SEQ ID NO:10.

The invention further provides a monoclonal antibody that competes forspecific binding to ApoE with 15G8 or a monoclonal antibody that bindsto the same epitope on ApoE as 15G8.

The invention further provides a humanized, chimeric or veneered form ofmonoclonal antibody 12C12, 15E10 or 15G8. Optionally, the antibodycomprises a light chain variable region comprising three light chainKabat CDRs of SEQ ID NO:18 and a heavy chain variable region comprisingthree heavy chain CDRs of SEQ ID NO:17. In some antibodies, the lightchain variable region framework has at least 85%, 90%, 91%, 92% 93%,94%, 95%, 96%, 97%, 98%, or 99% sequence identity to AAT86035 (SEQ IDNO:8). In some antibodies, the heavy chain variable region framework hasat least 85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or 99%sequence identity to AAX82494 (SEQ ID NO:7).

The invention further provides an antibody comprising a mature heavychain variable region having at least 85%, 90%, 91%, 92% 93%, 94%, 95%,96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:17, and/or a maturelight chain variable region having at least 85%, 90%, 91%, 92% 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:18. In some ofsuch antibodies, any differences in CDRs of the mature heavy chainvariable region and mature light variable region from SEQ ID NOs. 52 and60 respectively reside in positions H60-H65. In some such antibodies,the mature heavy chain variable region comprises the three Kabat CDRs ofSEQ ID NO:17 and the mature light chain variable region comprises thethree Kabat CDRs of SEQ ID NO:18. In some such antibodies, the matureheavy chain variable region has at least 85%, 90%, 91%, 92% 93%, 94%,95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:17. In somesuch antibodies, the mature light chain variable region has at least85%, 90%, 91%, 92% 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequenceidentity to SEQ ID NO:18. In some antibodies, position L3 (Kabatnumbering) is occupied by L. In some antibodies, position L36 (Kabatnumbering) is occupied by Y. In some antibodies, position L46 (Kabatnumbering) is occupied by L. In some antibodies, position H1 (Kabatnumbering) is occupied by E. In some antibodies, position H3 (Kabatnumbering) is occupied by K. In some antibodies, position H5 (Kabatnumbering) is occupied by V. In some antibodies, position H42 (Kabatnumbering) is occupied by E. In some antibodies, position H83 (Kabatnumbering) is occupied by R. In some antibodies, the amino acid sequenceof the mature heavy chain variable region is SEQ ID NO:17 and the aminoacid sequence of the mature light chain variable region is SEQ ID NO:18provided that position L3 (Kabat numbering) can be occupied by V or L,position L36 (Kabat numbering) can be occupied by F or Y, position L46(Kabat numbering) can be occupied by R or L, position H1 (Kabatnumbering) can be occupied by Q or E, position H3 (Kabat numbering) canbe occupied by Q or K, position H5 (Kabat numbering) can be occupied byQ or V, position H42 (Kabat numbering) can be occupied by D or E, andposition H83 (Kabat numbering) can be occupied by K or R. In some suchantibodies, position L3 (Kabat numbering) is occupied by L, position L36(Kabat numbering) is occupied by Y, and position L46 (Kabat numbering)is occupied by L. In some such antibodies, position L3 (Kabat numbering)is occupied by V, position L36 (Kabat numbering) is occupied by F, andposition L46 (Kabat numbering) is occupied by R. In some suchantibodies, position H1 (Kabat numbering) is occupied by E, position H3(Kabat numbering) is occupied by K, position H5 (Kabat numbering) isoccupied by V, position H42 (Kabat numbering) is occupied by E, andposition H83 (Kabat numbering) is occupied by R. In some suchantibodies, position H1 (Kabat numbering) is occupied by Q, position H3(Kabat numbering) is occupied by Q, position H5 (Kabat numbering) isoccupied by Q, position H42 (Kabat numbering) is occupied by D, andposition H83 (Kabat numbering) is occupied by K.

The invention further provides an antibody comprising a humanized heavychain comprising the three Kabat CDRs of SEQ ID NO:17 and a humanizedlight chain comprising the three CDRs of SEQ ID NO:18. In someantibodies, the mature heavy chain variable framework region having65-85% identity to the corresponding heavy chain variable regionframework of SEQ ID NO:17, and the mature light chain variable frameworkregion having 65-85% identity to the corresponding light chain variableregion framework of SEQ ID NO:18. Optionally, position L3 (Kabatnumbering) is occupied by L, and/or position L36 (Kabat numbering) isoccupied by Y, and/or position L46 (Kabat numbering) is occupied by L,and/or position H1 (Kabat numbering) is occupied by E, and/or positionH3 (Kabat numbering) is occupied by K, and/or position H5 (Kabatnumbering) is occupied by V, and/or position H42 (Kabat numbering) isoccupied by E, and/or position H83 (Kabat numbering) is occupied by R.Optionally, position L3 (Kabat numbering) is occupied by V, and/orposition L36 (Kabat numbering) is occupied by F, and/or position L46(Kabat numbering) is occupied by R, and/or position H1 (Kabat numbering)is occupied by Q, and/or position H3 (Kabat numbering) is occupied by Q,and/or position H5 (Kabat numbering) is occupied by Q, and/or positionH42 (Kabat numbering) is occupied by D, and/or position H83 (Kabatnumbering) is occupied by K. Optionally, position L3 (Kabat numbering)is occupied by L, and/or position L36 (Kabat numbering) is occupied byY, and/or position L46 (Kabat numbering) is occupied by L, and/orposition H1 (Kabat numbering) is occupied by Q, and/or position H3(Kabat numbering) is occupied by Q, and/or position H5 (Kabat numbering)is occupied by Q, and/or position H42 (Kabat numbering) is occupied byD, and/or position H83 (Kabat numbering) is occupied by K. Optionally,position L3 (Kabat numbering) is occupied by V, and/or position L36(Kabat numbering) is occupied by F, and/or position L46 (Kabatnumbering) is occupied by R, and/or position H1 (Kabat numbering) isoccupied by E, and/or position H3 (Kabat numbering) is occupied by K,and/or position H5 (Kabat numbering) is occupied by V, and/or positionH42 (Kabat numbering) is occupied by E, and/or position H83 (Kabatnumbering) is occupied by R. In some antibodies, position L3 (Kabatnumbering) is occupied by L, position L36 (Kabat numbering) is occupiedby Y, position L46 (Kabat numbering) is occupied by L, position H1(Kabat numbering) is occupied by E, position H3 (Kabat numbering) isoccupied by K, position H5 (Kabat numbering) is occupied by V, positionH42 (Kabat numbering) is occupied by E, and position H83 (Kabatnumbering) is occupied by R. In some antibodies, position L3 (Kabatnumbering) is occupied by V, position L36 (Kabat numbering) is occupiedby F, position L46 (Kabat numbering) is occupied by R, position H1(Kabat numbering) is occupied by Q, position H3 (Kabat numbering) isoccupied by Q, position H5 (Kabat numbering) is occupied by Q, positionH42 (Kabat numbering) is occupied by D, and position H83 (Kabatnumbering) is occupied by K. In some antibodies, position L3 (Kabatnumbering) is occupied by L, position L36 (Kabat numbering) is occupiedby Y, position L46 (Kabat numbering) is occupied by L, position H1(Kabat numbering) is occupied by Q, position H3 (Kabat numbering) isoccupied by Q, position H5 (Kabat numbering) is occupied by Q, positionH42 (Kabat numbering) is occupied by D, and position H83 (Kabatnumbering) is occupied by K. In some antibodies, position L3 (Kabatnumbering) is occupied by V, position L36 (Kabat numbering) is occupiedby F, position L46 (Kabat numbering) is occupied by R, position H1(Kabat numbering) is occupied by E, position H3 (Kabat numbering) isoccupied by K, position H5 (Kabat numbering) is occupied by V, positionH42 (Kabat numbering) is occupied by E, and position H83 (Kabatnumbering) is occupied by R.

The invention further provides an antibody comprising the mature heavychain variable region having an amino acid sequence of SEQ ID NO: 11 orSEQ ID NO:17 and a mature light chain variable region having an aminoacid sequence of SEQ ID NO:14 or SEQ ID NO:18. In some antibodies, themature heavy chain variable region has an amino acid sequence of SEQ IDNO:11 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:14. In some antibodies, the mature heavy chainvariable region has an amino acid sequence of SEQ ID NO:17 and themature light chain variable region has an amino acid sequence of SEQ IDNO:18. In some antibodies, the mature heavy chain variable region has anamino acid sequence of SEQ ID NO:11 and the mature light chain variableregion has an amino acid sequence of SEQ ID NO:18. In some antibodies,the mature heavy chain variable region has an amino acid sequence of SEQID NO:17 and the mature light chain variable region has an amino acidsequence of SEQ ID NO:14.

In any of the above antibodies, the mature heavy chain variable regioncan be fused to a heavy chain constant region and the mature light chainconstant region can be fused to a light chain constant region.

In any of the above antibodies, the heavy chain constant region can be amutant form of natural human constant region which has reduced bindingto an Fcγ receptor relative to the natural human constant region.

In any of the above antibodies, the heavy chain constant region can beof human IgG1 isotype. In some antibodies the allotype is Glm3. In someantibodies, the allotype is Glm1.

In some antibodies, the heavy chain constant region has the amino acidsequence designated SEQ ID NO:37, 38, 39, or 40 provided the C-terminallysine residue may be omitted. In some antibodies, the light chainconstant region has the amino acid sequence designated SEQ ID NO:35. Insome antibodies, the mature heavy chain variable region is fused to aheavy chain constant region having the amino acid sequence designatedSEQ ID NO:39 provided the C-terminal lysine residue may be omitted andthe mature light chain constant region is fused to a light chainconstant region having the amino acid sequence designated SEQ ID NO:35.In some antibodies, the mature light chain comprises SEQ ID NO:16 andthe mature heavy chain comprises SEQ ID NO:13. In some antibodies, themature heavy chain variable region has the amino acid sequence of SEQ IDNO:11, the heavy chain constant region has the amino acid sequence ofSEQ ID NO:39, the mature light chain variable region has the amino acidsequence of SEQ ID NO:14 and the light chain constant region has theamino acid sequence of SEQ ID NO:35.

The invention further provides a nucleic acid encoding any of theabove-mentioned mature heavy chain variable regions and/or any of theabove-mentioned mature light chain variable region, e.g., SEQ ID NO:12or SEQ ID NO:15.

The invention further provides a host cell comprising a vectorcomprising any of the nucleic acids described above.

The invention further provides a pharmaceutical composition comprisingany of the above-mentioned antibodies.

The invention further provides a method of humanizing an antibody,comprising determining the sequences of the heavy and light chainvariable regions of a mouse antibody, synthesizing a nucleic acidencoding a humanized heavy chain comprising CDRs of the mouse heavychain and a nucleic acid encoding a humanized light chain comprisingCDRs of the mouse light chain and expressing the nucleic acids in a hostcell to produce a humanized antibody, wherein the mouse antibody is12C12, 15E10 or 15G8.

The invention further provides a method of producing a humanized,chimeric or veneered antibody, comprising culturing cells transformedwith nucleic acids encoding the heavy and light chains of the antibody,so that the cells secrete the antibody; and purifying the antibody fromcell culture media; wherein the antibody is a humanized, chimeric orveneered form of 12C12, 15E10 or 15G8.

The invention further provides a method of producing a cell lineproducing a humanized, chimeric or veneered antibody, comprisingintroducing a vector encoding heavy and light chains of an antibody anda selectable marker into cells, propagating the cells under conditionsto select for cells having increased copy number of the vector,isolating single cells from the selected cell, and banking cells clonedfrom a single cell selected based on yield of antibody; wherein theantibody is a humanized, chimeric or veneered form of 12C12, 15E10 or15G8. Optionally, the method further comprises propagating the cellsunder selective conditions and screening for cell lines naturallyexpressing and secreting at least 100 pg/L/day.

The invention further provides an isolated antibody that specificallybinds to ApoE(1-299) at an epitope including residues 272 and 273.

The invention further provides a pharmaceutical composition comprisingany of the above antibodies and a pharmaceutically acceptable carrier.

The invention further provides an isolated fragment of ApoE including3-10 contiguous residues of ApoE and having a C-terminus ending atresidue 272. Optionally, the isolated fragment is linked to a carriermolecule optionally via a spacer that helps elicit antibodies againstthe fragment. The fragment can be combined with an adjuvant acceptablefor administration to humans to form a pharmaceutical composition.

The invention further provides a method of treating or effectingprophylaxis of Alzheimer's disease comprising administering an effectiveregime of an antibody that preferentially binds to ApoE(1-272) relativeto ApoE(1-299), or an agent that induces such an antibody, to a patienthaving or at risk of Alzheimer's disease and thereby treating oreffecting prophylaxis of the disease. Any of the antibodies describedabove can be used in such methods.

In some methods, the antibody comprises a mature heavy chain variableregion comprising the three Kabat CDRs of SEQ ID NO:9, and having atleast 90% sequence identity to SEQ ID NO:9, and a mature light chainvariable region comprising the three Kabat CDRs of SEQ ID NO:10, andhaving at least 90% sequence identity to SEQ ID NO:10. In some methods,the antibody is a humanized 15G8 antibody comprising a mature lightchain variable region framework having at least 85% sequence identity toAAT86035 (SEQ ID NO:8). In some methods, the antibody is a humanized15G8 antibody comprising a mature heavy chain variable region frameworkhaving at least 85% sequence identity to AAX82494 (SEQ ID NO:7). In somemethods, the antibody comprises a mature heavy chain variable regionhaving at least 90% sequence identity to SEQ ID NO:17, and a maturelight chain variable region having at least 90% sequence identity to SEQID NO:18. In some methods, the antibody comprises a mature heavy chainvariable region having an amino acid sequence of SEQ ID NO:17 and amature light chain variable region having an amino acid sequence of SEQID NO:18 provided that position L3 (Kabat numbering) can be occupied byV or L, position L36 (Kabat numbering) can be occupied by F or Y,position L46 (Kabat numbering) can be occupied by R or L, position H1(Kabat numbering) can be occupied by Q or E, position H3 (Kabatnumbering) can be occupied by Q or K, position H5 (Kabat numbering) canbe occupied by Q or V, position H42 (Kabat numbering) can be occupied byD or E, and position H83 (Kabat numbering) can be occupied by K or R. Insome methods, the antibody comprises a mature heavy chain variableregion having an amino acid sequence of SEQ ID NO:17 and a mature lightchain variable region having an amino acid sequence of SEQ ID NO:18provided that position L3 (Kabat numbering) is occupied by L, positionL36 (Kabat numbering) is occupied by Y, and position L46 (Kabatnumbering) is occupied by L. In some methods, the antibody comprises amature heavy chain variable region having an amino acid sequence of SEQID NO:17 and a mature light chain variable region having an amino acidsequence of SEQ ID NO:18 provided that position H1 (Kabat numbering) isoccupied by E, position H3 (Kabat numbering) is occupied by K, positionH5 (Kabat numbering) is occupied by V, position H42 (Kabat numbering) isoccupied by E, and position H83 (Kabat numbering) is occupied by R. Insome methods, the antibody comprises a mature heavy chain variableregion having an amino acid sequence of SEQ ID NO:11 and a mature lightchain variable region having an amino acid sequence of SEQ ID NO:14. Insome methods, the antibody comprises a mature heavy chain variableregion having the amino acid sequence of SEQ ID NO:11, a heavy chainconstant region having the amino acid sequence of SEQ ID NO:39, a maturelight chain variable region having the amino acid sequence of SEQ IDNO:14 and a light chain constant region having the amino acid sequenceof SEQ ID NO:35. Alternatively, a fragment of 3-10 contiguous aminoacids of ApoE with a C-terminus at residue 272 can be used. Optionally,such methods are performed on a patient who is an ApoE4 carrier.

The invention further provides a method of treating or effectingprophylaxis of a disease associated with ApoE4 comprising administeringan effective regime of an antibody that preferentially binds toApoE(1-272) relative to ApoE(1-299), or an agent that induces such anantibody, to a patient having or at risk of the disease and therebytreating or effecting prophylaxis of the disease.

The invention further provides a method of treating or effectingprophylaxis of neurological disease comprising administering aneffective regime of an antibody that preferentially binds to ApoE(1-272)relative to ApoE(1-299) or an agent that induces such an antibody to apatient who is an ApoE4 carrier and thereby treating or effectingprophylaxis of the disease.

The invention further provides a method of screening an agent foractivity against Alzheimer's disease, comprising administering the agentto a transgenic animal expressing ApoE and amyloid beta precursor (APP)transgenes, and determining whether the agent inhibits or delays atleast one sign or symptom of Alzheimer's disease, wherein the agent isan antibody that preferentially binds to ApoE(1-272) relative to ApoE(1-299) or the agent induces such an antibody.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an alignment of the amino acid sequences of mouse 15G8 withthe humanized 15G8 heavy chain mature variable region. The humanacceptor VH sequence from AAX82494 (GI: 62421461) (SEQ ID NO:7) is usedas human acceptor VH sequence.

FIG. 2 shows an alignment of the amino acid sequences of mouse 15G8 withthe humanized 15G8 light chain mature variable region. The humanacceptor VL sequence from AAT86035 (SEQ ID NO:8) is used as humanacceptor VL sequence.

FIG. 3 shows immunoprecipitation blots of full-length ApoE3, full-lengthApoE4, ApoE3(1-272), and ApoE4(1-272).

FIG. 4 shows western blot of tissue lysates from wild-type mouse (“wt”),apoE knockout mouse (“KO”), and transgenic mice that express human ApoEknock-in (E2, E3, and E4 strains) and human amyloid beta usingmonoclonal antibodies 12C12 (left panel), 15E10 (middle panel), and 15G8(right panel).

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is the ApoE3 amino acid sequence.

SEQ ID NO:2 is the ApoE4 amino acid sequence.

SEQ ID NO:3 is the ApoE3 (1-272) amino acid sequence.

SEQ ID NO:4 is the ApoE4 (1-272) amino acid sequence.

SEQ ID NO:5 is ApoE 268-272 with an artificial CGG added as linker andfor ease of coupling.

SEQ ID NO:6 is the amino acid sequence of ApoE4 (266-276).

SEQ ID NO:7 is the human acceptor V_(H) sequence from AAX82494.

SEQ ID NO:8 is the human acceptor V_(L) sequence from AAT86035.

SEQ ID NO:9 is the amino acid sequence of m15G8VH variable region (sameas m12C12VH or m15E10VH).

SEQ ID NO:10 is m15G8VL variable region (same as m12C12VL or m15E10VL).

SEQ ID NO:11 is the amino acid sequence of Hu15G8VHv1 variable region.

SEQ ID NO:12 is the nucleic acid sequence of Hu15G8VHv1 variable region.

SEQ ID NO:13 is the amino acid sequence of Hu15G8VHv1 fused with IgG1human Glm3 allotype constant region.

SEQ ID NO:14 is the amino acid sequence of Hu15G8VLv1 variable region.

SEQ ID NO:15 is the nucleic acid sequence of Hu15G8VLv1 variable region.

SEQ ID NO:16 is the amino acid sequence of Hu15G8VLv1 fused with humanlight chain kappa constant region (with Arginine at the N-terminal ofthe constant region).

SEQ ID NO:17 is the amino acid sequence of Hu15G8VHv2 variable regionhaving no backmutation in the variable framework region.

SEQ ID NO:18 is the amino acid sequence of Hu15G8VLv2 variable regionhaving no backmutation in the variable framework region.

SEQ ID NO:19 is the amino acid sequence of heavy chain Kabat CDR1.

SEQ ID NO:20 is the amino acid sequence of heavy chain Kabat CDR2.

SEQ ID NO:21 is the amino acid sequence of heavy chain Kabat CDR3.

SEQ ID NO:22 is the amino acid sequence of light chain Kabat CDR1.

SEQ ID NO:23 is the amino acid sequence of light chain Kabat CDR2.

SEQ ID NO:24 is the amino acid sequence of light chain Kabat CDR3.

SEQ ID NO:25 is the amino acid sequence of m15G8VH signal peptide.

SEQ ID NO:26 is the amino acid sequence of m12C12VH signal peptide.

SEQ ID NO:27 is the amino acid sequence of m15E10VH signal peptide.

SEQ ID NO:28 is the amino acid sequence of m15G8VL signal peptide (sameas m12C12VL or m15E10VL signal peptides).

SEQ ID NO:29 the nucleic acid sequence of Hu15G8VH signal peptide v1.

SEQ ID NO:30 the amino acid sequence of Hu15G8VH signal peptide v1.

SEQ ID NO:31 the nucleic acid sequence of Hu15G8VH signal peptide v2.

SEQ ID NO:32 the amino acid sequence of Hu15G8VH signal peptide v2.

SEQ ID NO:33 the nucleic acid sequence of Hu15G8VL signal peptide.

SEQ ID NO:34 the amino acid sequence of Hu15G8VL signal peptide.

SEQ ID NO:35 the amino acid sequence of human light chain kappa constantregion with arginine at the N-terminal.

SEQ ID NO:36 the amino acid sequence of human light chain kappa constantregion without arginine at the N-terminal.

SEQ ID NO:37 the amino acid sequence of human IgG1 heavy chain constantregion.

SEQ ID NO:38 the amino acid sequence of human IgG1 Glm1 allotype heavychain constant region.

SEQ ID NO:39 the amino acid sequence of human IgG1 Glm3 allotype heavychain constant region.

SEQ ID NO:40 is the amino acid sequence of human IgG2 heavy chainconstant region.

SEQ ID NO:41 is ApoE 268-272.

DEFINITIONS

Monoclonal antibodies and other therapeutic agents are typicallyprovided in isolated form. This means that the agent is typically atleast 50% w/w pure of interfering proteins and other contaminantsarising from its production or purification but does not exclude thepossibility that the agent is combined with an excess of pharmaceuticalacceptable carrier(s) or other vehicle intended to facilitate its use.Sometimes monoclonal antibodies are at least 60%, 70%, 80%, 90%, 95% or99% w/w pure of interfering proteins and contaminants from production orpurification.

Antibodies of the invention typically bind to their designated targetwith an association constant of at least 10⁶, 10⁷, 10⁸, 10⁹, or 10¹⁰ M.Such binding is specific binding in that it is detectably higher inmagnitude and distinguishable from non-specific binding occurring to atleast one unrelated target. Specific binding can be the result offormation of bonds between particular functional groups or particularspatial fit (e.g., lock and key type) whereas nonspecific binding isusually the result of van der Waals forces. Specific binding does nothowever necessarily imply that a monoclonal antibody binds one and onlyone target.

The basic antibody structural unit is a tetramer of subunits. Eachtetramer includes two identical pairs of polypeptide chains, each pairhaving one “light” (about 25 kDa) and one “heavy” chain (about 50-70kDa). The amino-terminal portion of each chain includes a variableregion of about 100 to 110 or more amino acids primarily responsible forantigen recognition, e.g., a variable light chain region (VL) and avariable heavy chain region (VH). This variable region is initiallyexpressed linked to a cleavable signal peptide. The variable regionwithout the signal peptide is sometimes referred to as a mature variableregion. Thus, for example, a light chain mature variable region, means alight chain variable region without the light chain signal peptide. Thecarboxy-terminal portion of each chain defines a constant regionprimarily responsible for effector function.

Light chains are classified as either kappa or lambda. Heavy chains areclassified as gamma, mu, alpha, delta, or epsilon, and define theantibody's isotype as IgG, IgM, IgA, IgD and IgE, respectively. Withinlight and heavy chains, the variable and constant regions are joined bya “J” region of about 12 or more amino acids, with the heavy chain alsoincluding a “D” region of about 10 or more amino acids. (See generally,Fundamental Immunology (Paul, W., ed., 2nd ed. Raven Press, N.Y., 1989),Ch. 7) (incorporated by reference in its entirety for all purposes).

The mature variable regions of each light/heavy chain pair form theantibody binding site. Thus, an intact antibody has two binding sites.Except in bifunctional or bispecific antibodies, the two binding sitesare the same. The chains all exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions, also called complementarity determining regions(CDRs). The CDRs from the two chains of each pair are aligned by theframework regions, enabling binding to a specific epitope. FromN-terminal to C-terminal, both light and heavy chains comprise thedomains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The assignment of aminoacids to each domain is in accordance with the definitions of Kabat,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md., 1987 and 1991), or Chothia & Lesk, J. Mol. Biol.196:901-917 (1987); Chothia et al., Nature 342:878-883 (1989). Kabatalso provides a widely used numbering convention (Kabat numbering) inwhich corresponding residues between different heavy chains or betweendifferent light chains are assigned the same number.

The term “antibody” includes intact antibodies and binding fragmentsthereof. Typically, fragments compete with the intact antibody fromwhich they were derived for specific binding to the target. Fragmentsinclude separate heavy chains, light chains Fab, Fab′, F(ab)₂, F(ab)c,Fv and single domain antibodies. Single (variable) domain antibodiesinclude VH regions separated from their VL partners (or vice versa) inconventional antibodies (Ward et al., 1989, Nature 341: 544-546) as wellas VH regions (sometimes known as VHH) from species such as Camelidae orcartilaginous fish (e.g., a nurse shark) in which VH regions are notassociated with VL regions (see, e.g., WO 9404678). The former type ofantibodies are sometimes known as Dabs and the latter are sometimesknown as nanobodies. Constant regions or parts of constant regions mayor may not be present in single domain antibodies. For example, naturalsingle variable domain antibodies from Camelidae include a VHH variableregion, and CH2 and CH3 constant regions. Single domain antibodies canbe subject of humanization by analogous approaches to conventionalantibodies. The Dabs type of antibodies are usually obtained fromantibodies of human origin. Nanobody™ types of antibody are of Camelidaeor shark origin and can be subject to humanization. Fragments can beproduced by recombinant DNA techniques, or by enzymatic or chemicalseparation of intact immunoglobulins. As well as monospecificantibodies, the term “antibody” also includes a bispecific antibody. Abispecific or bifunctional antibody is an artificial hybrid antibodyhaving two different heavy/light chain pairs and two different bindingsites (see, e.g., Songsivilai and Lachmann, Clin. Exp. Immunol.79:315-321 (1990); Kostelny et al., J. Immunol., 148:1547-53 (1992)). Insome bispecific antibodies, the two different heavy/light chain pairsinclude a monoclonal or humanized 15G8 heavy chain/light chain pair anda heavy chain/light chain pair specific for a different epitope on ApoEthan that bound by 15G8.

In some bispecific antibodies, one heavy chain/light chain pair is amonoclonal or humanized 15G8 antibody as further disclosed below and theheavy/light chain pair is from an antibody that binds to a receptorexpressed on the blood brain barrier, such as an insulin receptor, aninsulin-like growth factor (IGF) receptor, a leptin receptor, alipoprotein receptor, or a transferrin receptor (Friden et al., PNAS88:4771-4775, 1991; Friden et al., Science 259:373-377, 1993). Such abispecific antibody can be transferred cross the blood brain barrier byreceptor-mediated transcytosis. Brain uptake of the bispecific antibodycan be further enhanced by engineering the bi-specific antibody toreduce its affinity to the blood brain barrier receptor. Reducedaffinity for the receptor has resulted in a broader distribution in thebrain (see, e.g., Atwal. et al. Sci. Trans. Med. 3, 84ra43, 2011; Yu etal. Sci. Trans. Med. 3, 84ra44, 2011).

The term “epitope” refers to a site on an antigen to which an antibodybinds. An epitope can be formed from contiguous amino acids ornoncontiguous amino acids juxtaposed by tertiary folding of one or moreproteins. Epitopes formed from contiguous amino acids are typicallyretained on exposure to denaturing solvents whereas epitopes formed bytertiary folding are typically lost on treatment with denaturingsolvents. An epitope typically includes at least 3, and more usually, atleast 5 or 8-10 amino acids in a unique spatial conformation. Methods ofdetermining spatial conformation of epitopes include, for example, x-raycrystallography and 2-dimensional nuclear magnetic resonance. See, e.g.,Epitope Mapping Protocols, in Methods in Molecular Biology, Vol. 66,Glenn E. Morris, Ed. (1996).

Antibodies that recognize the same or overlapping epitopes can beidentified in a simple immunoassay showing the ability of one antibodyto compete with the binding of another antibody to a target antigen. Theepitope of an antibody can also be defined by X-ray crystallography ofthe antibody bound to its antigen to identify contact residues.Alternatively, two antibodies have the same epitope if all amino acidmutations in the antigen that reduce or eliminate binding of oneantibody reduce or eliminate binding of the other. Two antibodies haveoverlapping epitopes if some amino acid mutations that reduce oreliminate binding of one antibody reduce or eliminate binding of theother.

Competition between antibodies is determined by an assay in which anantibody under test inhibits specific binding of a reference antibody toa common antigen (see, e.g., Junghans et al., Cancer Res. 50:1495,1990). A test antibody competes with a reference antibody if an excessof a test antibody (e.g., at least 2×, 5×, 10×, 20× or 100×) inhibitsbinding of the reference antibody by at least 50% but preferably 75%,90% or 99% as measured in a competitive binding assay. Antibodiesidentified by competition assay (competing antibodies) includeantibodies binding to the same epitope as the reference antibody andantibodies binding to an adjacent epitope sufficiently proximal to theepitope bound by the reference antibody for steric hindrance to occur.

The term “patient” includes human and other mammalian subjects thatreceive either prophylactic or therapeutic treatment.

For purposes of classifying amino acids substitutions as conservative ornonconservative, amino acids are grouped as follows: Group I(hydrophobic side chains): met, ala, val, leu, ile; Group II (neutralhydrophilic side chains): cys, ser, thr; Group III (acidic side chains):asp, glu; Group IV (basic side chains): asn, gln, his, lys, arg; Group V(residues influencing chain orientation): gly, pro; and Group VI(aromatic side chains): tip, tyr, phe. Conservative substitutionsinvolve substitutions between amino acids in the same class.Non-conservative substitutions constitute exchanging a member of one ofthese classes for a member of another.

Percentage sequence identities are determined with antibody sequencesmaximally aligned by the Kabat numbering convention. After alignment, ifa subject antibody region (e.g., the entire mature variable region of aheavy or light chain) is being compared with the same region of areference antibody, the percentage sequence identity between the subjectand reference antibody regions is the number of positions occupied bythe same amino acid in both the subject and reference antibody regiondivided by the total number of aligned positions of the two regions,with gaps not counted, multiplied by 100 to convert to percentage.

The term “adjuvant” refers to a compound that when administered inconjunction with an antigen augments and/or redirects the immuneresponse to the antigen, but when administered alone does not generatean immune response to the antigen. Adjuvants can augment an immuneresponse by several mechanisms including lymphocyte recruitment,stimulation of B and/or T cells, and stimulation of macrophages.

For brevity, the term “ApoE4 carrier” is sometimes used to refer topatients having one or two ApoE4 alleles and “ApoE4 noncarrier”, “ApoE4non-carrier” or “non-ApoE4 carrier” to refer to patients having zeroApoE4 alleles.

A disease is associated with the ApoE4 allele if a population of ApoE4carriers has a greater frequency of occurrence of the disease, greaterseverity of the disease or earlier age of onset of the disease comparedwith a population of non-carriers to a statistically significant extent.

An individual is at increased risk of a disease if the subject has atleast one known risk-factor (e.g., genetic, biochemical, family history,situational exposure) placing individuals with that risk factor at astatistically significant greater risk of developing the disease thanindividuals without the risk factor.

The term “symptom” refers to subjective evidence of a disease, such asaltered gait, as perceived by the patient. A “sign” refers to objectiveevidence of a disease as observed by a physician.

Statistical significance means p≦0.05.

DETAILED DESCRIPTION OF THE INVENTION I. General

Normal human ApoE is 299 amino acids in length not including a signalpeptide but can be truncated by cleavage with a chymotrypsin-like serineprotease in vivo to generate C-terminal truncations and particularly afragment truncated at amino acid 272. ApoE4 more readily undergoesproteolytic cleavage than other ApoE isoforms. The 1-272 fragment ofApoE4 is neurotoxic in tissue culture and in transgenic mice expressingthe 1-272 fragment.

The present invention provides antibodies that preferentially bind to anApoE(1-272) fragment relative to ApoE(1-299) or relative to otherC-terminally truncated form of ApoE terminating at residue 273 orgreater. These antibodies serve to reduce the toxicity of this fragment.Although an understanding of mechanism is not required for practice ofthe invention, a reduction in toxicity may occur as a result of theantibody inducing phagocytosis of the fragment, or reducing the freeconcentration of ApoE(1-272), or inhibiting the fragment from inter orintramolecular aggregation, or from binding to other molecules, or bystabilizing a non-toxic conformation among other mechanisms. Because theantibody preferentially binds to ApoE(1-272) over full-length ApoE, thetoxicity of truncated fragments can be inhibited without unacceptablereduction of the neuroprotective role of full length ApoE(1-299).

Antibodies that preferentially bind to an ApoE(1-272) fragment or agentsthat can induce such antibodies can be used in methods of treating oreffecting prophylaxis of Alzheimer's disease and other diseasesassociated with the presence of ApoE4.

II. ApoE and Fragments Thereof.

Unless otherwise apparent from the context, ApoE refers to a naturalhuman form of ApoE, particularly the ApoE2, E3 or E4 allele thereof. Anexemplary sequence of the E3 allele (residues 19-317 of Swiss-ProtPO₂₆₄₉) is provided below.

(SEQ ID NO: l)KVEQAVETEP EPELRQQTEW QSGQRWELAL GRFWDYLRWV QTLSEQVQEE LLSSQVTQEL RALMDETMKELKAYKSELEE QLTPVAEETR ARLSKELQAA QARLGADMED VCGRLVQYRG EVQAMLGQST EELRVRLASHLRKLRKRLLR DADDLQKRLA VYQAGAREGA ERGLSAIRER LGPLVEQGRV RAATVGSLAG QPLQERAQAWGERLRARMEE MGSRTRDRLD EVKEQVAEVR AKLEEQAQQI RLQAEAFQAR LKSWFEPLVE DMQRQWAGLVEKVQAAVGTS AAPVPSDNH

The underlined cysteine at position 112 is arginine in ApoE4 and theunderlined arginine at position 158 is cysteine in ApoE2. Thus, ApoE4has the following sequence

(SEQ ID NO: 2)KVEQAVETEP EPELRQQTEW QSGQRWELAL GRFWDYLRWV QTLSEQVQEE LLSSQVTQEL RALMDETMKELKAYKSELEE QLTPVAEETR ARLSKELQAA QARLGADMED VRGRLVQYRG EVQAMLGQST EELRVRLASHLRKLRKRLLR DADDLQKRLA VYQAGAREGA ERGLSAIRER LGPLVEQGRV RAATVGSLAG QPLQERAQAWGERLRARMEE MGSRTRDRLD EVKEQVAEVR AKLEEQAQQI RLQAEAFQAR LKSWFEPLVE DMQRQWAGLVEKVQAAVGTS AAPVPSDNH

Fragments of ApoE are sometimes referred to by providing a range of thefirst and last amino acid, as for example ApoE(1-272). As for the fulllength ApoE protein, such a fragment can include any of the E2, E3 or E4alleles. A preferred fragment is ApoE(1-272). The E3 and E4 alleles ofthis fragment are shown below, with E3 first.

(SEQ ID NO: 3)KVEQAVETEP EPELRQQTEW QSGQRWELAL GRFWDYLRWV QTLSEQVQEE LLSSQVTQEL RALMDETMKELKAYKSELEE QLTPVAEETR ARLSKELQAA QARLGADMED VCGRLVQYRG EVQAMLGQST EELRVRLASHLRKLRKRLLR DADDLQKRLA VYQAGAREGA ERGLSAIRER LGPLVEQGRV RAATVGSLAG QPLQERAQAWGERLRARMEE MGSRTRDRLD EVKEQVAEVR AKLEEQAQQI RLQAEAFQAR LKSWFEPLVE DM(SEQ ID NO: 4)KVEQAVETEP EPELRQQTEW QSGQRWELAL GRFWDYLRWV QTLSEQVQEE LLSSQVTQEL RALMDETMKELKAYKSELEE QLTPVAEETR ARLSKELQAA QARLGADMED VRGRLVQYRG EVQAMLGQST EELRVRLASHLRKLRKRLLR DADDLQKRLA VYQAGAREGA ERGLSAIRER LGPLVEQGRV RAATVGSLAG QPLQERAQAWGERLRARMEE MGSRTRDRLD EVKEQVAEVR AKLEEQAQQI RLQAEAFQAR LKSWFEPLVE DM

The allelic forms present in any individual can be determined by manyconventional techniques, such as direct sequencing, use of GeneChip®arrays or the like, allele-specific probes, single-base extensionmethods, allelic specific extension. Allelic forms can also bedetermined at the protein level by ELISA using antibodies specific fordifferent allelic expression products. Kits for genetic andimmunological analysis are commercially available (e.g., Innogenetics,Inc.). Determination of allelic forms are usually made in vitro, thatis, on samples removed and never returned to a patient.

III. Antibodies A. Binding Specificity and Functional Properties

The invention provides antibodies preferentially binding to ApoE(1-272)relative to ApoE(1-299). The antibodies can be monoclonal or polyclonal.Antibodies designated 12C12, 15E10 or 15G8 are three exemplary mousemonoclonal antibodies of IgG1k isotype. The mouse monoclonal antibodies12C12, 15E10, and 15G8 have the same mature variable regions. The matureheavy chain variable region of mouse monoclonal 12C12, 15E10, or 15G8 isSEQ ID NO:9. The mature light chain variable region of mouse monoclonal12C12, 15E10 or 15G8 is SEQ ID NO:10.

> m15G8VH variable region (same as m12C12VH or m15E10VH) (SEQ ID NO: 9)EVKLVESGGGLVKPGGSLKLSCAASGFTFS

WVRQTPEKRLEWVA

RFTISRDNARNTLYLQMSSLRSEDTAMFYCAR

WGQGTSVTV SS (SEQ ID NO: 10) >m15G8VL variable region (same as m12C12VL or m15E10VL)DVLMTQTPLSLPVSLGDQASISC

WYLQKPGQSPKLLIY

GVPDRFSGSGSGTDFTLKISRVEAEDLGIYYC

FGGGTKLEIK

Preferential binding means that an antibody binds to ApoE(1-272)detectably more strongly (i.e., higher association constant) than toApoE(1-299) (i.e., beyond experimental error). Preferred antibodies haveassociation constants at least 2, 5 or 10-fold higher for ApoE(1-272)than ApoE(1-299). Some antibodies bind to ApoE(1-272) and lack anysignificant binding to ApoE(1-299) (i.e., binding indistinguishablebetween ApoE(1-299) and an irrelevant control protein). Some antibodiespreferentially binding to ApoE(1-272) over ApoE(1-299) are end-specificfor the free C-terminus of ApoE(1-272). Such antibodies recognize anepitope including the C-terminal amino acid of ApoE(1-272) in free form(i.e., with a carboxyl group not attached to another amino acid asoccurs in ApoE(1-299)). End-specific antibodies can bind for example toan epitope within residues 263-272, 264-272, 265-272, 266-272, 267-272,268-272, 269-272 or 270-272 of ApoE (within being inclusive of thepositions used to define the range). End-specific antibodies toApoE(1-272) show preferential binding for ApoE(1-272) relative toApoE(1-299) but may or may not lack any degree of specific binding toApoE(1-299). Other antibodies preferentially binding to ApoE(1-272) overApoE(1-299) are not end-specific but may recognize a conformationalepitope present on ApoE(1-272) that is not present or at least notprecisely replicated in ApoE(1-299) due for example to differences infolding patterns between ApoE(1-272) and ApoE(1-299).

End-specific antibodies to the C-terminus of ApoE(1-272) can begenerated de novo by immunizing with a peptide including the C-terminalamino acid of this fragment. Usually, such peptides have 3-10 contiguousamino acids from the C-terminus including the C-terminal amino acid,with peptides of 5 or 6 contiguous amino acids being preferred. Suchpeptides are preferably attached to a heterologous conjugate moleculethat helps elicit an antibody response to the peptide. Attachment can bedirect or via a spacer peptide or amino acid. Cysteine is used as aspacer amino acid because its free SH group facilitates attachment of acarrier molecule. A polyglycine linker (e.g., 2-6 glycines), with orwithout a cysteine residue between the glycines and the peptide can alsobe used. The carrier molecule serves to provide a T-cell epitope thathelps elicit an antibody response against the peptide. Several carrierscan be used, including keyhole limpet hemocyanin (KLH), ovalbumin andbovine serum albumin (BSA). Peptide spacers can be added to the peptideimmunogen as part of solid phase peptide synthesis. Peptide spacers canbe added to peptide immunogen as part of solid phase peptide synthesis.Carriers are typically added by chemical cross-linking. Some examples ofchemical crosslinkers that can be used includecross-N-maleimido-6-aminocaproyl ester orm-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) (see for example,Harlow, E. et al., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. 1988; Sinigaglia et al.,Nature, 336:778-780 (1988); Chicz et al., J. Exp. Med., 178:27-47(1993); Hammer et al., Cell 74:197-203 (1993); Falk K. et al.,Immunogenetics, 39:230-242 (1994); WO 98/23635; and, Southwood et al. J.Immunology, 160:3363-3373 (1998)). Because the goal is to generateantibodies to the free C-terminus of the peptide, the carrier and spacerif present are typically attached to the N-terminus of the peptide.Exemplary protocols for generating end-specific antibodies against otherpeptides are described by e.g., Konig, Ann NY Acad Sci 777:344-355(1996), Harrington, Biochim. Biophys. Acta 1158 (2):120-128 (1993);Gravina et al., J. Biol. Chem. 270:(13):7013-6 (1995).

A peptide with optional spacer and carrier can be used to immunize alaboratory animals or B-cells as described in more detail below.Hybridoma supernatants can be tested for ability to bind the ApoEpeptide used as an immunogen. The peptide can be attached to a carrieror other tag to facilitate the screening assay. In this case, thecarrier or tag is preferentially different than the combination ofspacer and carrier molecule used for immunization to eliminateantibodies specific for the spacer or carrier rather than the ApoEpeptide. Antibodies can also be screened against a peptide bridging thesite of truncation of the peptide immunogen. For example, if the peptideimmunogen ends at residue 272 of ApoE, a peptide bridging the site oftruncation includes at least residues 272 and 273 of ApoE. Antibodiescan also be screened for binding to ApoE(1-272) and for lack of bindingor at least reduced binding to ApoE(1-299). The ApoE used in such assayscan be any of the E2, E3 or E4 isoforms.

Some antibodies of the invention bind to the same or overlapping epitopeas mouse monoclonal antibodies 12C12, 15E10 or 15G8. Some antibodiescompete for specific binding to ApoE with a mouse monoclonal antibody12C12, 15E10 or 15G8. Antibodies having such a binding epitope orspecificity can be produced by immunizing mice with ApoE or a portionthereof including the desired epitope, and screening resultingantibodies for preferentially binding to ApoE(1-272) relative toApoE(1-299), optionally in competition with a mouse monoclonal antibody12C12, 15E10 or 15G8.

Antibodies having the binding specificity of a selected murine antibody(e.g. 12C12, 15E10 or 15G8) can also be produced using a variant of thephage display method. See Winter, WO 92/20791. This method isparticularly suitable for producing human antibodies. In this method,either the heavy or light chain variable region of the selected murineantibody is used as a starting material. If, for example, a light chainvariable region is selected as the starting material, a phage library isconstructed in which members display the same light chain variableregion (i.e., the murine starting material) and a different heavy chainvariable region. The heavy chain variable regions can for example beobtained from a library of rearranged human heavy chain variableregions. A phage showing strong specific binding for ApoE(1-272) (e.g.,at least 10⁸ and preferably at least 10⁹ M⁻¹) is selected. The heavychain variable region from this phage then serves as a starting materialfor constructing a further phage library. In this library, each phagedisplays the same heavy chain variable region (i.e., the regionidentified from the first display library) and a different light chainvariable region. The light chain variable regions can be obtained forexample from a library of rearranged human variable light chain regions.Again, phage showing strong specific binding for Apo E(1-272) areselected. The resulting antibodies usually have the same or similarepitope specificity as the murine starting material.

Other antibodies can be obtained by mutagenesis of cDNA encoding theheavy and light chains of an exemplary antibody, such as 12C12, 15E10 or15G8. Monoclonal antibodies that are at least 62.5%, 65%, 75%, 85%, 90%,95% or 99% identical to 12C12, 15E10 or 15G8 in amino acid sequence ofthe mature heavy and/or light chain variable regions and maintain itsfunctional properties, and/or which differ from the respective antibodyby a small number of functionally inconsequential amino acidsubstitutions (e.g., conservative substitutions), deletions, orinsertions are also included in the invention. In some such antibodies,each CDR differs in sequence from a corresponding CDR from 15G8 by nomore than 6 (e.g., 1, 2, 3, 4, 5, or 6) replacements, deletions orinsertions. Monoclonal antibodies having at least one and preferably allsix CDR(s) as defined by Kabat that are at least 62.5%, 65%, 75%, 85%,90%, 95%, 99% or 100% identical to corresponding CDRs of 12C12, 15E10 or15G8 are also included. Some antibodies have at least 80, 95 or 100%identity to each CDR of 12C12, 15E10 or 15G8 other than CDR H2 and atleast 62.5% identity to CDR H2. The mouse monoclonal antibodies 12C12,15E10, and 15G8 each have three Kabat light chain CDRs of SEQ ID NO:22,SEQ ID NO:23, and SEQ ID NO:24, and three Kabat heavy chain CDRs of SEQID NO:19, SEQ ID NO:20, and SEQ ID NO:21.

heavy chain Kabat CDR1

(SEQ ID NO: 19) heavy chain Kabat CDR2

(SEQ ID NO: 20) heavy chain Kabat CDR3

(SEQ ID NO: 21) light chain Kabat CDR1

(SEQ ID NO: 22) light chain Kabat CDR2

(SEQ ID NO: 23) light chain CDR3 Kabat

(SEQ ID NO: 24)

Antibodies discriminating between ApoE(1-272) and ApoE(1-299) that arenot end-specific but bind to conformational epitopes present inApoE(1-272) but not present or not precisely replicated in ApoE(1-299)can be produced by immunizing with longer peptide immunogens sufficientto develop a characteristic conformation, for example ApoE(1-272) itselfor at least 50, 100, 200 or 250 contiguous residues thereof. Longerpeptides can be produced by recombinant expression among other methods.Antibodies generated by such methods are screened for preferentialbinding to ApoE(1-272) relative to ApoE(1-299).

The invention also provides antibodies that preferably bind to longertruncated fragments, i.e., fragments from ApoE(1-273) to ApoE(1-298)relative to ApoE(1-299) including antibodies that are end-specific forthe C-terminus of such fragments.

The invention also provides antibodies having an epitope including thetwo residues on either side of a site of proteolytic cleavage. For thesite of cleavage, generating ApoE(1-272), the two such residues areresidues 272 and 273 of ApoE. Such antibodies can be generated using apeptide as immunogen including the two residues on either side of thecleavage site. As in generating an end-specific antibody, such a peptidetypically includes about 3-10 contiguous amino acids from ApoEaltogether. The peptide can be linked to a conjugate molecule optionallyvia a spacer as described in producing end-specific antibodies.

B. Non-Human Antibodies

The production of other non-human monoclonal antibodies, e.g., murine,guinea pig, primate, rabbit or rat, against an immunogen can beperformed by, for example, immunizing the animal with an immunogen asdescribed above. See Harlow & Lane, Antibodies, A Laboratory Manual(CSHP NY, 1988) (incorporated by reference for all purposes). Such animmunogen can be obtained from a natural source, by peptide synthesis orby recombinant expression.

Optionally, the immunogen can be administered with an adjuvant. Severaltypes of adjuvant can be used as described below. Complete Freund'sadjuvant followed by incomplete adjuvant is preferred for immunizationof laboratory animals. Rabbits or guinea pigs are typically used formaking polyclonal antibodies. Mice are typically used for makingmonoclonal antibodies. Antibodies are screened for specific binding toApoE(1-272) or other truncated form of ApoE. Optionally, antibodies arefurther screened for lack of specific binding to ApoE(1-299) or to apeptide of ApoE including residues spanning residues 272-273 or apeptide spanning another cleavage site generating a truncated form ofApoE. Such screening can be accomplished by determining binding of anantibody to a collection of deletion mutants and determining whichdeletion mutants bind to the antibody. Binding can be assessed, forexample, by Western blot, FACS™ or ELISA.

C. Humanized Antibodies

A humanized antibody is a genetically engineered antibody in which theCDRs from a non-human “donor” antibody (e.g., 12C12, 15E10 or 15G8) aregrafted into human “acceptor” antibody sequences (see, e.g., Queen, U.S.Pat. Nos. 5,530,101 and 5,585,089; Winter, U.S. Pat. No. 5,225,539;Carter, U.S. Pat. No. 6,407,213; Adair, U.S. Pat. Nos. 5,859,205 and6,881,557, and Foote, U.S. Pat. No. 6,881,557). The acceptor antibodysequences can be, for example, a mature human antibody sequence, acomposite of such sequences, a consensus sequence of human antibodysequences (e.g., light or heavy chain variable region consensussequences of Kabat, 1991, supra), or a germline variable regionsequence.

An example of an acceptor sequence for the heavy chain is the humanmature heavy chain variable region with GenBank accession code AAX82494(GI: 62421461). CDR H1 and H2 of this acceptor sequence are of the samecanonical forms as those of mouse 15G8 heavy chain (CDR H3 of 15G8 hasno canonical class). This acceptor sequence has a sequence identity of82% in the heavy chain variable region framework to mouse 15G8 heavychain variable region framework. If a different acceptor sequence isused, such an acceptor can be, for example, a mature heavy chainvariable region derived from the human mature heavy chain variableregion with GenBank accession code AAL57837 (GI: 18042117; 77% sequenceidentity to mouse 15G8 heavy chain variable region framework) or amature heavy chain variable region sequence incorporating one of suchsequences.

For the light chain, an example of an acceptor sequence is the maturelight chain variable region with GenBank accession code AAT86035(GI:50898163). This acceptor sequence includes three CDRs having thesame canonical form as a mouse 15G8 light chain and has a sequenceidentity of 81% in the light chain variable region framework. If adifferent acceptor is used, such an acceptor can be, for example,another mature light chain sequence derived from the mature light chainvariable region with GenBank accession code BAC01730 (GI:2166941 1; 81%sequence identity to mouse 15G8 light chain variable region framework)or ABC66863 (GI: 84797828; 80% sequence identity to mouse 15G8 lightchain variable region framework) or a mature light chain variable regionsequence incorporating one of such sequences.

A humanized antibody is an antibody having some or all CDRs entirely orsubstantially from a donor antibody and variable region frameworksequences and constant regions, if present, entirely or substantiallyfrom human antibody sequences. Similarly a humanized heavy chain has atleast one, two and usually all three CDRs entirely or substantially froma donor antibody heavy chain, and a heavy chain variable regionframework sequence and heavy chain constant region, if present,substantially from human heavy chain variable region framework andconstant region sequences. Similarly a humanized light chain has atleast one, two and usually all three CDRs entirely or substantially froma donor antibody light chain, and a light chain variable regionframework sequence and light chain constant region, if present,substantially from human light chain variable region framework andconstant region sequences. Other than nanobodies and dAbs, a humanizedantibody comprises a humanized heavy chain and a humanized light chain.A CDR in a humanized antibody is substantially from a corresponding CDRin a non-human antibody when at least 85%, 90%, 95% or 100% ofcorresponding residues (as defined by Kabat) are identical between therespective CDRs. The variable region framework sequences of an antibodychain or the constant region of an antibody chain are substantially froma human variable region framework sequence or human constant regionrespectively when at least 85, 90, 95 or 100% of corresponding residuesdefined by Kabat are identical.

Although humanized antibodies often incorporate all six CDRs (preferablyas defined by Kabat) from a mouse antibody, they can also be made withless than all CDRs (e.g., at least 3, 4, or 5) CDRs from a mouseantibody (e.g., Pascalis et al., J. Immunol. 169:3076, 2002; Vajdos etal., Journal of Molecular Biology, 320: 415-428, 2002; Iwahashi et al.,Mol. Immunol. 36:1079-1091, 1999; Tamura et al, Journal of Immunology,164:1432-1441, 2000).

In some antibodies only part of the CDRs, namely the subset of CDRresidues required for binding, termed the SDRs, are needed to retainbinding in a humanized antibody. CDR residues not contacting antigen andnot in the SDRs can be identified based on previous studies (for exampleresidues H60-H65 in CDR H2 are often not required), from regions ofKabat CDRs lying outside Chothia hypervariable loops (Chothia, J. Mol.Biol. 196:901, 1987), by molecular modeling and/or empirically, or asdescribed in Gonzales et al., Mol. Immunol. 41: 863, 2004. Thus, forexample, some humanized antibodies can have a CDR H2 with up to 6replacements, deletions or insertions, or at least 62.5% sequenceidentity with CDR H2 of the donor antibody. In such humanized antibodiesat positions in which one or more donor CDR residues is absent or inwhich an entire donor CDR is omitted, the amino acid occupying theposition can be an amino acid occupying the corresponding position (byKabat numbering) in the acceptor antibody sequence. The number of suchsubstitutions of acceptor for donor amino acids in the CDRs to includereflects a balance of competing considerations. Such substitutions arepotentially advantageous in decreasing the number of mouse amino acidsin a humanized antibody and consequently decreasing potentialimmunogenicity. However, substitutions can also cause changes ofaffinity, and significant reductions in affinity are preferably avoided.Positions for substitution within CDRs and amino acids to substitute canalso be selected empirically.

The human acceptor antibody sequences can optionally be selected fromamong the many known human antibody sequences to provide a high degreeof sequence identity (e.g., 65-85% identity) between a human acceptorsequence variable region frameworks and corresponding variable regionframeworks of a donor antibody chain.

Certain amino acids from the human variable region framework residuescan be selected for substitution based on their possible influence onCDR conformation and/or binding to antigen. Investigation of suchpossible influences is by modeling, examination of the characteristicsof the amino acids at particular locations, or empirical observation ofthe effects of substitution or mutagenesis of particular amino acids.

For example, when an amino acid differs between a murine variable regionframework residue and a selected human variable region frameworkresidue, the human framework amino acid can be substituted by theequivalent framework amino acid from the mouse antibody when it isreasonably expected that the amino acid:

-   -   (1) noncovalently binds antigen directly,    -   (2) is adjacent to a CDR region,    -   (3) otherwise interacts with a CDR region (e.g. is within about        6 Å of a CDR region), (e.g., identified by modeling the light or        heavy chain on the solved structure of a homologous known        immunoglobulin chain); and    -   (4) a residue participating in the VL-VH interface.

Framework residues from classes (1)-(3) as defined by Queen, U.S. Pat.No. 5,530,101 are sometimes alternately referred to as canonical andvernier residues. Framework residues defining canonical class of thedonor CDR loops determining the conformation of a CDR loop are sometimesreferred to as canonical residues (Chothia and Lesk, J. Mol. Biol. 196,901-917 (1987), Thornton & Martin J. Mol. Biol., 263, 800-815, 1996). Alayer of framework residues that support antigen-binding loopconformations play a role in fine-tuning the fit of an antibody toantigen are sometimes referred to as vernier residues (Foote & Winter,1992, J Mol Bio. 224, 487-499). Other candidates for substitution areresidues creating a potential glycosylation site. Other candidates forsubstitution are acceptor human framework amino acids that are unusualfor a human immunoglobulin at that position. These amino acids can besubstituted with amino acids from the equivalent position of the mousedonor antibody or from the equivalent positions of more typical humanimmunoglobulins. Other candidates for substitution are acceptor humanframework amino acids that are unusual for a human immunoglobulin atthat position.

Exemplary humanized antibodies of the invention include a humanized formof 15G8, characterized by a mature light chain variable region of SEQ IDNO:14 or SEQ ID NO:18 (designated L1 and L2, respectively) and a matureheavy chain variable region of SEQ ID NO:11 or SEQ ID NO:17 (designatedH1 and H2, respectively). For example, humanized antibodies includeH1L1, H2L2, H1L2, and H2L1.

The invention also provides variants of H1L1, H2L2, H1L2, and H2L 1. Insuch variants, the humanized heavy chain mature variable region shows atleast 65%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQID NO:11 or SEQ ID NO:17 and the humanized light chain mature variableregion shows at least 65%, 85%, 90%, 93%, 94%, 95%, 96%, 97%, 98% or 99%sequence identity to SEQ ID NO:14 or SEQ ID NO:18. Some such humanizedantibodies include three heavy and three light chain Kabat CDRs entirelyor substantially identical to the Kabat CDR regions of H1L1, H2L2, H1L2,and H2L1, which are the same as those of the mouse donor antibody.

Some variants differ from the sequences of H1L1, H2L2, H1L2, or H2L1 bya small number (e.g., typically no more than 1, 2, 3, 5, 6, 7, 8, 9 or10) of replacements, deletions or insertions. Such differences areusually in the framework but can also occur in the CDRs. Many of theframework residues not in contact with the CDRs in the humanized mAb canaccommodate substitutions of amino acids from the correspondingpositions of the donor mouse mAb or other mouse or human antibodies, andeven many potential CDR-contact residues are also amenable tosubstitution or even amino acids within the CDRs may be altered. Oneexample of a CDR substitution is to substitute a residue in a CDR withthe residue occupying the corresponding position of the human acceptorsequence used to supply variable region frameworks.

Often the replacements made in the variants of H1L1, H2L2, H1L2, andH2L1 are conservative with respect to the replaced amino acids. In somevariants, replacements in H1L1, H2L2, H1L2, and H2L1 (whether or notconservative) have no substantial effect on the binding affinity of thehumanized antibody. In some variants, the mature variant light and heavychain variable region sequences are at least 90%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, or at least 98% identicalto the respective H1L1, H2L2, H1L2, and H2L1 mature light and heavychain variable regions. Alternatively, other human antibody acceptorsequences, particularly those with high sequence identity to thevariable region framework sequences of murine 15G8 are also suitable toprovide the humanized antibody variable regions framework sequences.

In some variants of H1L1, H2L2, H1L2, and H2L1, at least 1, 2, 3, 4, 5,6, 7 or all 8 of the positions of acceptor to donor substitutionsmentioned in connection with the exemplified antibody (i.e., L3, L36,L46, H1, H3, H5, H42, and H83) are occupied by residues L, Y, L, E, K,V, E, and R respectively (the residues occupying the correspondingposition of the mouse donor antibody heavy chain). If the heavy chainacceptor sequence is other than AAX82494, or the light chain acceptorsequence is other than AAT86035 an acceptor to donor substitution may ormay not be required for the specified occupancy of a particular variableframework region position depending on whether the residue occupying thespecified position is already the same between the acceptor and donor.

D. Chimeric and Veneered Antibodies

The invention further provides chimeric and veneered forms of non-humanantibodies, particularly the 12C12, 15E10 or 15G8 antibodies of theexamples.

A chimeric antibody is an antibody in which the mature variable regionsof light and heavy chains of a non-human antibody (e.g., a mouse) arecombined with human light and heavy chain constant regions. Suchantibodies substantially or entirely retain the binding specificity ofthe mouse antibody, and are about two-thirds human sequence.

A veneered antibody is a type of humanized antibody that retains someand usually all of the CDRs and some of the non-human variable regionframework residues of a non-human antibody but replaces other variableregion framework residues that may contribute to B- or T-cell epitopes,for example exposed residues (Padlan, Mol. Immunol. 28:489, 1991) withresidues from the corresponding positions of a human antibody sequence.The result is an antibody in which the CDRs are entirely orsubstantially from a non-human antibody and the variable regionframeworks of the non-human antibody are made more human-like by thesubstitutions.

E. Human Antibodies.

Human antibodies against ApoE(1-272) or other truncated form of ApoE areprovided by a variety of techniques described below. Methods forproducing human antibodies include the trioma method of Oestberg et al.,Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664; andEngleman et al., U.S. Pat. No. 4,634,666, use of transgenic miceincluding human immunoglobulin genes (see, e.g., Lonberg et al.,WO93/12227 (1993); U.S. Pat. No. 5,877,397, U.S. Pat. No. 5,874,299,U.S. Pat. No. 5,814,318, U.S. Pat. No. 5,789,650, U.S. Pat. No.5,770,429, U.S. Pat. No. 5,661,016, U.S. Pat. No. 5,633,425, U.S. Pat.No. 5,625,126, U.S. Pat. No. 5,569,825, U.S. Pat. No. 5,545,806, Nature148, 1547-1553 (1994), Nature Biotechnology 14, 826 (1996),Kucherlapati, WO 91/10741 (1991) and phage display methods (see, e.g.Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047, U.S. Pat.No. 5,877,218, U.S. Pat. No. 5,871,907, U.S. Pat. No. 5,858,657, U.S.Pat. No. 5,837,242, U.S. Pat. No. 5,733,743 and U.S. Pat. No. 5,565,332.

F. Selection of Constant Region

The heavy and light chain variable regions of chimeric, humanized(including veneered), or human antibodies can be linked to at least aportion of a human constant region. The choice of constant regiondepends, in part, whether antibody-dependent complement and/or cellularmediated cytotoxicity is desired. For example, human isotopes IgG1 andIgG3 have complement-mediated cytotoxicity and human isotypes IgG2 andIgG4 do not. Light chain constant regions can be lambda or kappa.

An exemplary human light chain kappa constant region has the amino acidsequence of SEQ ID NO:35. The N-terminal arginine of SEQ ID NO:35 can beomitted, in which case light chain kappa constant region has the aminoacid sequence of SEQ ID NO:36. An exemplary human IgG1 heavy chainconstant region has the amino acid sequence of SEQ ID NO:37. Anexemplary human IgG2 heavy chain constant region has the amino acidsequence of SEQ ID NO:38. Antibodies can be expressed as tetramerscontaining two light and two heavy chains, as separate heavy chains,light chains, as Fab, Fab′, F(ab′)2, and Fv, or as single chainantibodies in which heavy and light chain variable domains are linkedthrough a spacer.

Human constant regions show allotypic variation and isoallotypicvariation between different individuals, that is, the constant regionscan differ in different individuals at one or more polymorphicpositions. Isoallotypes differ from allotypes in that sera recognizingan isoallotype binds to a non-polymorphic region of a one or more otherisotypes. Reference to a human constant region includes a constantregion with any natural allotype or any permutation of residuesoccupying polymorphic positions in natural allotypes or up to 3, 5 or 10substitutions for reducing or increasing effector function as describedbelow. For example, heavy chain constant regions can be of IgG1 Glm1 orIgG1 Glm3 allotypes and have the amino acid sequence of SEQ ID NO:38 orSEQ ID NO:39, respectively. Yet another heavy chain constant region hasthe amino acid sequence of SEQ ID NO:38 or SEQ ID NO:39 except that itlacks the C-terminal lysine.

One or several amino acids at the amino or carboxy terminus of the lightand/or heavy chain, such as the C-terminal lysine of the heavy chain,may be missing or derivatized in a proportion or all of the molecules.Substitutions can be made in the constant regions to reduce or increaseeffector function such as complement-mediated cytotoxicity or ADCC (see,e.g., Winter et al., U.S. Pat. No. 5,624,821; Tso et al., U.S. Pat. No.5,834,597; and Lazar et al., Proc. Natl. Acad. Sci. USA 103:4005, 2006),or to prolong half-life in humans (see, e.g., Hinton et al., J. Biol.Chem. 279:6213, 2004). Exemplary substitutions include a Gln at position250 and/or a Leu at position 428 (EU numbering is used in this paragraphfor the constant region) for increasing the half life of an antibody.Substitution at any or all of positions 234, 235, 236 and/or 237 reduceaffinity for Fcγ receptors, particularly FcγRI receptor (see, e.g., U.S.Pat. No. 6,624,821). An alanine substitution at positions 234, 235 and237 of human IgG1 is preferred for reducing effector functions.Optionally, positions 234, 236 and/or 237 in human IgG2 are substitutedwith alanine and position 235 with glutamine (See, e.g., U.S. Pat. No.5,624,821).

In some antibodies, the light chain constant region has the amino acidsequence of SEQ ID NO:35. In some antibodies, the heavy chain constantregion has the amino acid sequence of SEQ ID NO:39. An exemplaryhumanized light chain has an amino acid sequence of SEQ ID NO:16. Anexemplary humanized heavy chain has the amino acid sequence of SEQ IDNO:13.

> Hu15G8VHv2 fused with IgG1 human G1m3 allotype constant region(SEQ ID NO: 13)EVKLVESGGGLVKPGGSLKLSCAASGFTFSFYAMSWVRQTPEKRLEWVASLSRGGSTYYPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCAREGATALYAMDYWGQGTMVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK >Hu15G8VLv1 fused with human light chain kappa constant region(with Arginine at the N-terminal of the constant region) (SEQ ID NO: 16)DVLMTQSPLSLPVTLGQPASISCRSSQSIVHSNGNTYLQWYLQRPGQSPRLLLYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

G. Expression of Recombinant Antibodies

Chimeric, humanized (including veneered) and human antibodies aretypically produced by recombinant expression. Nucleic acids encoding theantibodies can be codon-optimized for expression in the desiredcell-type (e.g., CHO or Sp2/0). Recombinant polynucleotide constructstypically include an expression control sequence operably linked to thecoding sequences of antibody chains, including naturally-associated orheterologous promoter regions. Preferably, the expression controlsequences are eukaryotic promoter systems in vectors capable oftransforming or transfecting eukaryotic host cells. Once the vector hasbeen incorporated into the appropriate host, the host is maintainedunder conditions suitable for high level expression of the nucleotidesequences, and the collection and purification of the crossreactingantibodies. The vector or vectors encoding the antibody chains can alsocontain a selectable gene, such as dihydrofolate reductase, to allowamplification of copy number of the nucleic acids encoding the antibodychains.

E. coli is a prokaryotic host particularly useful for expressingantibodies, particularly antibody fragments. Microbes, such as yeast arealso useful for expression. Saccharomyces is a preferred yeast host,with suitable vectors having expression control sequences, an origin ofreplication, termination sequences and the like as desired. Typicalpromoters include 3-phosphoglycerate kinase and other glycolyticenzymes. Inducible yeast promoters include, among others, promoters fromalcohol dehydrogenase, isocytochrome C, and enzymes responsible formaltose and galactose utilizations

Mammalian cells are a preferred host for expressing nucleotide segmentsencoding immunoglobulins or fragments thereof. See Winnacker, From Genesto Clones, (VCH Publishers, NY, 1987). A number of suitable host celllines capable of secreting intact heterologous proteins have beendeveloped in the art, and include CHO cell lines, various COS celllines, HeLa cells, HEK293 cells, L cells, and non-antibody-producingmyelomas including Sp2/0 and NSO. Preferably, the cells are nonhuman.Expression vectors for these cells can include expression controlsequences, such as an origin of replication, a promoter, an enhancer(Queen et al., Immunol. Rev. 89:49 (1986)), and necessary processinginformation sites, such as ribosome binding sites, RNA splice sites,polyadenylation sites, and transcriptional terminator sequences.Preferred expression control sequences are promoters derived fromendogenous genes, cytomegalovirus, SV40, adenovirus, bovinepapillomavirus, and the like. See Co et al., J. Immunol. 148:1149(1992).

Having introduced vector(s) encoding antibody heavy and light chainsinto cell culture, cell pools can be screened for growth productivityand product quality in serum-free media. Top-producing cell pools canthen be subjected of FACS-based single-cell cloning to generatemonoclonal lines. Specific productivites above 50 pg or 100 pg per cellper day, which correspond to product titers of greater than 7.5 g/Lculture, are preferred. Antibodies produced by single cell clones canalso be tested for turbidity, filtration properties, PAGE, IEF, UV scan,HP-SEC, carboydrate-oligosaccharide mapping, mass spectrometery, andbining assay, such as ELISA or Biacore. A selected clone can then bebanked in multiple vials and stored frozen for subsequent use.

Once expressed, antibodies can be purified according to standardprocedures of the art, including protein A capture, columnchromatography (e.g., hydrophobic interaction or ion exchange), low-pHfor viral inactivation and the like (see generally, Scopes, ProteinPurification (Springer-Verlag, NY, 1982)).

Methodology for commercial production of antibodies including codonoptimization, selection of promoters, transcription elements, andterminators, serum-free single cell cloning, cell banking, use ofselection markers for amplification of copy number, CHO terminator,serum free single cell cloning, improvement of protein titers (see,e.g., U.S. Pat. No. 5,786,464, U.S. Pat. No. 5,888,809, U.S. Pat. No.6,063,598, U.S. Pat. No. 6,114,148, U.S. Pat. No. 7,569,339,WO2004/050884, WO2005/019442, WO2008/012142, WO2008/107388, andWO2009/027471).

H. Nucleic Acids

The invention further provides nucleic acids encoding any of the heavyand light chains described above. Typically, a nucleic acid also encodesa signal peptide fused to the mature heavy or light chain encoded by thenucleic acid (e.g., signal peptides having amino acid sequences of SEQID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28; signal peptideshaving amino acid sequences of SEQ ID NO:30, SEQ ID NO:32, and SEQ IDNO:34 that can be encoded by SEQ ID NO:29, SEQ ID NO:31, and SEQ IDNO:33). Coding sequences on nucleic acids can be in operable linkagewith regulatory sequences to ensure expression of the coding sequences,such as a promoter, enhancer, ribosome binding site, transcriptiontermination signal and the like. The nucleic acids encoding heavy andlight chains can occur in isolated form or can be cloned into one ormore vectors. The nucleic acids can be synthesized by for example, solidstate synthesis or PCR of overlapping oligonucleotides. Nucleic acidsencoding heavy and light chains can be joined as one contiguous nucleicacid, e.g., within an expression vector, or can be separate, e.g., eachcloned into its own expression vector.

IV. Active Immunogens

An agent used for active immunization serves to induce in a patient thesame types of antibody described in connection with passive immunizationabove (e.g., an antibody preferentially binding to ApoE(1-272) or othertruncated form of ApoE over ApoE(1-299)). Agents used for activeimmunization can be the same types of immunogens used for generatingmonoclonal antibodies in laboratory animals (e.g., a peptide of 3-10contiguous amino acids from the C-terminus of a desired truncatedfragment, such as ApoE(1-272). Some examples of fragments that can beused include ApoE 270-272, 269-272, 268-272, 267-272, 266-272, 265-272,264-272, and 263-272 with 268-272 and 267-272 being preferred.

The heterologous carrier and adjuvant, if used may be the same as usedfor generating monoclonal antibody, but may also be selected for betterpharmaceutical suitability for use in humans. Suitable carriers includeserum albumins, keyhole limpet hemocyanin, immunoglobulin molecules,thyroglobulin, ovalbumin, tetanus toxoid, or a toxoid from otherpathogenic bacteria, such as diphtheria (e.g., CRM197), E. coli,cholera, or H. pylori, or an attenuated toxin derivative. T cellepitopes are also suitable carrier molecules. Some conjugates can beformed by linking agents of the invention to an immunostimulatorypolymer molecule (e.g., tripalmitoyl-S-glycerine cysteine (Pam₃Cys),mannan (a mannose polymer), or glucan (a β1→2 polymer)), cytokines(e.g., IL-1, IL-1 alpha and β peptides, IL-2, γ-INF, IL-10, GM-CSF), andchemokines (e.g., MIP1-α and β, and RANTES). Immunogens may be linked tothe carriers with or without spacers amino acids (e.g., gly-gly).Additional carriers include virus-like particles. Virus-like particles(VLPs), also called pseudovirions or virus-derived particles, representsubunit structures composed of multiple copies of a viral capsid and/orenvelope protein capable of self assembly into VLPs of defined sphericalsymmetry in vivo. (Powilleit, et al., (2007) PLoS ONE 2(5):e415.)Alternatively, peptide immunogens can be linked to at least oneartificial T-cell epitope capable of binding a large proportion of MHCClass II molecules, such as the pan DR epitope (“PADRE”). PADRE isdescribed in U.S. Pat. No. 5,736,142, WO 95/07707, and Alexander J etal, Immunity, 1:751-761 (1994). Active immunogens can be presented inmultimeric form in which multiple copies of an immunogen and/or itscarrier are presented as a single covalent molecule.

Fragments are often administered with pharmaceutically acceptableadjuvants. The adjuvant increases the titer of induced antibodies and/orthe binding affinity of induced antibodies relative to the situation ifthe peptide were used alone. A variety of adjuvants can be used incombination with an immunogenic fragment of ApoE, to elicit an immuneresponse. Preferred adjuvants augment the intrinsic response to animmunogen without causing conformational changes in the immunogen thataffect the qualitative form of the response. Preferred adjuvants includealuminum salts, such aluminum hydroxide and aluminum phosphate, 3De-O-acylated monophosphoryl lipid A (MPL™) (see GB 2220211 (RIBIImmunoChem Research Inc., Hamilton, Mont., now part of Corixa).Stimulon™ QS-21 is a triterpene glycoside or saponin isolated from thebark of the Quillaja Saponaria Molina tree found in South America (seeKensil et al., in Vaccine Design: The Subunit and Adjuvant Approach(eds. Powell & Newman, Plenum Press, NY, 1995); U.S. Pat. No.5,057,540), (Aquila BioPharmaceuticals, Framingham, Mass.; nowAntigenics, Inc., New York, N.Y.). Other adjuvants are oil in wateremulsions (such as squalene or peanut oil), optionally in combinationwith immune stimulants, such as monophosphoryl lipid A (see Stoute etal., N. Engl. J. Med. 336, 86-91 (1997)), pluronic polymers, and killedmycobacteria. Another adjuvant is CpG (WO 98/40100). Adjuvants can beadministered as a component of a therapeutic composition with an activeagent or can be administered separately, before, concurrently with, orafter administration of the therapeutic agent.

Analogs of natural fragments of ApoE that induce antibodies againstApoE(1-272) can also be used. For example, one or more or all L-aminoacids can be substituted with D amino acids in such peptides. Also theorder of amino acids can be reversed (retro peptide). Optionally apeptide includes all D-amino acids in reverse order (retro-inversopeptide). Peptides and other compounds that do not necessarily have asignificant amino acid sequence similarity with ApoE peptides butnevertheless serve as mimetics of ApoE peptides and induce a similarimmune response. Anti-idiotypic antibodies against monoclonal antibodiesto ApoE as described above can also be used. Such anti-Id antibodiesmimic the antigen and generate an immune response to it (see EssentialImmunology, Roit ed., Blackwell Scientific Publications, Palo Alto,Calif. 6th ed., p. 181).

Peptides (and optionally a carrier fused to the peptide) can also beadministered in the form of a nucleic acid encoding the peptide andexpressed in situ in a patient. A nucleic acid segment encoding animmunogen is typically linked to regulatory elements, such as a promoterand enhancer that allow expression of the DNA segment in the intendedtarget cells of a patient. For expression in blood cells, as isdesirable for induction of an immune response, promoter and enhancerelements from light or heavy chain immunoglobulin genes or the CMV majorintermediate early promoter and enhancer are suitable to directexpression. The linked regulatory elements and coding sequences areoften cloned into a vector.

The DNA can be delivered in naked form (i.e., without colloidal orencapsulating materials). Alternatively a number of viral vector systemscan be used including retroviral systems (see, e.g., Lawrie and Tumin,Cur. Opin. Genet. Develop. 3, 102-109 (1993)); adenoviral vectors (see,e.g., Bett et al, J. Virol. 67, 591 1 (1993)); adeno-associated virusvectors (see, e.g., Zhou et al., J. Exp. Med. 179, 1867 (1994)), viralvectors from the pox family including vaccinia virus and the avian poxviruses, viral vectors from the alpha virus genus such as those derivedfrom Sindbis and Semliki Forest Viruses (see, e.g., Dubensky et al., J.Virol. 70, 508-519 (1996)), Venezuelan equine encephalitis virus (seeU.S. Pat. No. 5,643,576) and rhabdoviruses, such as vesicular stomatitisvirus (see WO 96/34625) and papillomaviruses (Ohe et al., Human GeneTherapy 6, 325-333 (1995); Woo et al, WO 94/12629 and Xiao & Brandsma,Nucleic Acids. Res. 24, 2630-2622 (1996)).

DNA encoding an immunogen, or a vector containing the same, can bepackaged into liposomes. Suitable lipids and related analogs aredescribed by U.S. Pat. No. 5,208,036, U.S. Pat. No. 5,264,618, U.S. Pat.No. 5,279,833, and U.S. Pat. No. 5,283,185. Vectors and DNA encoding animmunogen can also be adsorbed to or associated with particulatecarriers, examples of which include polymethyl methacrylate polymers andpolylactides and poly(lactide-co-glycolides), (see, e.g., McGee et al.,J. Micro Encap. 1996).

V. Screening Methods

Antibodies can be initially screened for the intended bindingspecificity as has already been described (e.g., preferential binding toApoE(1-272) over ApoE(1-299)). Active immunogens can likewise bescreened for capacity to induce antibodies with such bindingspecificity. In this case, an active immunogen is used to immunize alaboratory animal and the resulting sera tested for the appropriatebinding specificity.

Antibodies having the desired binding specificity can then be tested incellular and animal models. Cellular models include cells naturallyexpressing ApoE or transfected with DNA encoding ApoE(1-299) or atruncated fragment thereof, particularly ApoE(1-272). The cells used forsuch screening are preferentially neuronal cells. Cells can be screenedfor reduced levels of ApoE(1-272) (e.g., by Western blotting orimmunoprecipitation of cell extracts) or reduced toxicity attributableto ApoE(1-272) as described in the Examples. Antibodies or activeimmunogens can also be screened in transgenic animal models of diseasesassociated with ApoE4. Such transgenic animals can include a human ApoEand/or human APP transgene among others, such as tau, presenilin oralpha synuclein. Such transgenic animals are disposed to develop atleast one sign or symptom of a disease associated with ApoE. Numeroustransgenic mice with human ApoE transgenes have been described in thescientific literature, varying in e.g., the allele expressed, knock-outof endogenous alleles, full-length ApoE transgene or a truncatedfragment (e.g., ApoE(1-272)), selection of promoter, among other factors(see world wide web alzforum.org/res/com/tra), Buttini et al., J.Neurosci. 22, 10539-10548 (2002), Holtzman et al., PNAS 97, 2892-2897(2000), Tesseur et al., Am. J. Path. 157, 1495-1510 (2000) Raffai etal., Circulation 102: 11-150 (abstr.) (2000); Raffai et al., J. Biol.Chem. 277: 11064-11068 (2002). Features of Alzheimer's pathology inthese models include reduced numbers of presynaptic terminals, increasedplaque deposition (in models expressing both ApoE and APP), increasedtau phosphorylation, impaired learning and memory and altered long termpotentiation, significant learning impairment that can be assessed usinga water maze (see Marley et al., PNAS 103, 5644-5651 (2006)). Theactivity of antibodies or active agents can be assessed by variouscriteria including reduction in ApoE truncated fragments, particularlyApoE(1-272), reduction in other pathological characteristics, such asamyloid deposits of Aβ, and inhibition or delay or behavioral deficits.Active immunogens can also be tested for induction of antibodies in thesera. Both passive and active immunogens can be tested for passage ofantibodies across the blood brain barrier into the brain of a transgenicanimal. Tests on an antibody or active agent are usually performed inconjunction with a control in which a parallel experiment is conductedexcept that the antibody or active agent is absent (e.g., replaced byvehicle). Any of the following ApoE4 antibodies (15D2, 12D3, 7C8 and2G3), ApoE3 antibodies (12E5, 12H5 and 6H6), 2C11 (recognizes both ApoE3and ApoE4) and 5F6 (recognizes ApoE but not otherwise characterized)among others can be used as controls. Reduction, delay or inhibition ofsigns or symptoms disease attributable to an antibody or active agentunder test can then be assessed relative to the control.

VI. Patients Amenable to Treatment

The presence of an ApoE4 allele has been associated with increased risk,increased severity and/or earlier age of onset of a large number ofneurological disease and conditions including Alzheimer's disease,Down's syndrome, mild cognitive impairment, vascular amyloid disease(e.g., cerebral amyloid angiopathy), Parkinson's disease and other Lewybody disorders, heat trauma, stroke, complications of coronary bypasssurgery, amyotrophic lateral sclerosis, multiple sclerosis, diabeticneuropathy, sleep disorders, and CNS ischemia (see, e.g., Mayley et al.,PNAS 103, 5644-5651 (2006)). Because of the widespread associationbetween neurological diseases and conditions and an ApoE4 allele, thepresent regimes can be used in treatment or prophylaxis of any subjectthat is carrier of an ApoE4 allele having any neurological disease orconsidered at risk of developing one. The present regimes can also beused for treatment or prophylaxis in any of the above-mentioned diseasesor others associated with an ApoE4 allele or elevated levels ofApoE1-272 on individuals regardless of ApoE4 carrier status. The presentmethods are particularly suitable for treatment or prophylaxis ofAlzheimer's disease, and especially in patients who are ApoE4 carriers.

Patients amenable to treatment include individuals at risk of diseasebut not showing symptoms, as well as patients presently showingsymptoms. Patients at risk of disease include those having a knowngenetic risk of a disease. Such individuals include those havingrelatives who have experienced this disease, and those whose risk isdetermined by analysis of genetic or biochemical markers. Geneticmarkers of risk include particularly the ApoE4 allele in heterozygousand even more so in homozygous form. Other markers of risk ofAlzheimer's disease include mutations in the APP gene, particularlymutations at position 717 and positions 670 and 671 referred to as theHardy and Swedish mutations respectively, mutations in the presenilingenes, PS1 and PS2, a family history of AD, hypercholesterolemia oratherosclerosis. Individuals presently suffering from Alzheimer'sdisease can be recognized by PET imaging, from characteristic dementia,as well as the presence of risk factors described above. In addition, anumber of diagnostic tests are available for identifying individuals whohave AD. These include measurement of CSF tau and A1342 levels. Elevatedtau and decreased A1342 levels signify the presence of AD. Somemutations associated with Parkinson's disease. Ala30Pro or Ala53, ormutations in other genes associated with Parkinson's disease such asleucine rich repeat kinase, PARK8. Individuals can also be diagnosedwith any of the neurological diseases mentioned above by the criteria ofthe DSM IV TR.

In asymptomatic patients, treatment can begin at any age (e.g., 10, 20,30). Usually, however, it is not necessary to begin treatment until apatient reaches 40, 50, 60 or 70 years of age. Treatment typicallyentails multiple dosages over a period of time. Treatment can bemonitored by assaying antibody levels over time. If the response falls,a booster dosage is indicated. In the case of potential Down's syndromepatients, treatment can begin antenatally by administering therapeuticagent to the mother or shortly after birth.

VII. Pharmaceutical Compositions and Methods of Treatment

In prophylactic applications, an antibody or agent for inducing anantibody or a pharmaceutical composition the same is administered to apatient susceptible to, or otherwise at risk of a disease (e.g.,Alzheimer's disease) in regime (dose, frequency and route ofadministration) effective to reduce the risk, lessen the severity, ordelay the onset of at least one sign or symptom of the disease. Inparticular, the regime is preferably effective to inhibit or delayaccumulation of ApoE(1-272) in the brain, and/or inhibit or delay itstoxic effects and/or inhibit/or delay development of behavioraldeficits. In therapeutic applications, an antibody or agent to induce anantibody is administered to a patient suspected of, or already sufferingfrom a disease (e.g., Alzheimer's disease) in a regime (dose, frequencyand route of administration) effective to ameliorate or at least inhibitfurther deterioration of at least one sign or symptom of the disease. Inparticular, the regime is preferably effective to reduce or at leastinhibit further increase of levels of ApoE(1-272), associated toxicitiesand/or behavioral deficits.

A regime is considered therapeutically or prophylactically effective ifan individual treated patient achieves an outcome more favorable thanthe mean outcome in a control population of comparable patients nottreated by methods of the invention, or if a more favorable outcome isdemonstrated in treated patients versus control patients in a controlledclinical trial (e.g., a phase II, phase II/III or phase III trial) atthe p<0.05 or 0.01 or even 0.001 level.

Effective doses of vary depending upon many different factors, includingmeans of administration, target site, physiological state of thepatient, whether the patient is an ApoE carrier, whether the patient ishuman or an animal, other medications administered, and whethertreatment is prophylactic or therapeutic.

An exemplary dosage range for antibodies is from about 0.01 to 5 mg/kg,and more usually 0.1 to 3 mg/kg or 0.15-2 mg/kg or 0.15-1.5 mg/kg, ofpatient body weight. Antibody can be administered such doses daily, onalternative days, weekly, fortnightly, monthly, quarterly, or accordingto any other schedule determined by empirical analysis. An exemplarytreatment entails administration in multiple dosages over a prolongedperiod, for example, of at least six months. Additional exemplarytreatment regimes entail administration once per every two weeks or oncea month or once every 3 to 6 months.

The amount of an agent for active administration varies from 0.1-500 μgper patient and more usually from 1-100 or 1-10 μg per injection forhuman administration. The timing of injections can vary significantlyfrom once a day, to once a year, to once a decade. A typical regimenconsists of an immunization followed by booster injections at timeintervals, such as 6 week intervals or two months. Another regimenconsists of an immunization followed by booster injections 1, 2 and 12months later. Another regimen entails an injection every two months forlife. Alternatively, booster injections can be on an irregular basis asindicated by monitoring of immune response.

Antibodies or agents for inducing antibodies are preferably administeredvia a peripheral route (i.e., one in which an administered or inducedantibody crosses the blood brain barrier to reach an intended site inthe brain. Routes of administration include topical, intravenous, oral,subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal,intranasal or intramuscular. Preferred routes for administration ofantibodies are intravenous and subcutaneous. Preferred routes for activeimmunization are subcutaneous and intramuscular. This type of injectionis most typically performed in the arm or leg muscles. In some methods,agents are injected directly into a particular tissue where depositshave accumulated, for example intracranial injection.

Pharmaceutical compositions for parenteral administration are preferablysterile and substantially isotonic and manufactured under GMPconditions. Pharmaceutical compositions can be provided in unit dosageform (i.e., the dosage for a single administration). Pharmaceuticalcompositions can be formulated using one or more physiologicallyacceptable carriers, diluents, excipients or auxiliaries. Theformulation depends on the route of administration chosen. Forinjection, antibodies can be formulated in aqueous solutions, preferablyin physiologically compatible buffers such as Hank's solution, Ringer'ssolution, or physiological saline or acetate buffer (to reducediscomfort at the site of injection). The solution can containformulatory agents such as suspending, stabilizing and/or dispersingagents. Alternatively antibodies can be in lyophilized form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The present regimes can be administered in combination with anotheragent effective in treatment or prophylaxis of the disease beingtreated. For example, in the case of Alzheimer's disease, the presentregimes can be combined with immunotherapy against Aβ (WO/2000/072880),cholinesterase inhibitors or memantine or in the case of Parkinson'sdisease immunotherapy against alpha synuclein WO/2008/103472, Levodopa,dopamine agonists, COMT inhibitors, MAO-B inhibitors, Amantadine, oranticholinergic agents.

EXAMPLES Example 1 Antibody Preparation

To prepare antibodies specific for the neo-epitope of ApoE ending at 272CGG-LVEDM (SEQ ID NO:5); ApoE 268-272 (LVEDM; SEQ ID NO:41) with anartificial CGG added as linker and for ease of coupling) was conjugatedto Sheep anti Mouse IgG(H+ L) using EMCS([N-e-Maleimidocaproyloxy]succinimide ester) which allows thecross-linking of the free amines on the sheep anti mouse to the cysteineon the peptide.

Five A/J mice were immunized on day 0 with 100 μg of the peptideconjugate in Freund's Complete Adjuvant, and again on day 14, 28 and 56with 100 μg of peptide in Freund's Incomplete Adjuvant. On day 63 micewere bled via a tail vein nick and dilutions of serum were then used todetermine antibody titers to the CGG-LVEDM peptide (SEQ ID NO:5). Allanimals raised an acceptable titer.

Animal #5 had a titer of 218000 and was chosen for fusion. Fusion wasdone using a modification of the method of Kohler and Milstein. Fusedcells were incubated overnight in selection media, viable cells countedand resuspended in CloneMedia for Hybridomas from Genetix atapproximately 5000 cells/well with the addition of azaserine forselection of fused cells, mIL6 to promote growth, and CGG-LVEDM (SEQ IDNO:5) coupled to activated ovalbumin then fluorescently labeled to imagepositive clones.

Initial selection was done using ClonePix-FL software. Colonies deemedpositive by our criteria were transferred to 96 well plates, allowed togrow to 50-80% confluence and rescreened for binding to CGG-LVEDM (SEQID NO:5) and no binding to a peptide that spans aa 266-276 of ApoE(EPLVEDMQRQW; SEQ ID NO:6). Antibodies that met this criteria and showedgood stability were cloned and expanded to purify the antibody fromtissue culture media.

Antibodies can also be tested for binding to ApoE(1-272). TheApoE(1-272) fragment can be any of the ApoE2, E3 or E4 isoforms. Such afragment is preferably recombinantly expressed. Binding can be assessedby immunoprecipitation and/or western blots. Antibodies can also betested by immunohistochemistry on AD brain tissue from Apo E2, E3 or E4carriers compared with controls to see if a given antibody is specificfor the appropriate fragments.

Example 2 In Vitro Testing of Antibodies

Neuro 2A cells (mouse neuroblast from neuroblastoma, ATCC; Cat. No.CCL-131) expressing transfected human ApoE4 process it to the 1-272fragment which exerts neurotoxicity at a number of levels (as detectedby a simple 3-(4,5-demithylthiazol-2-yl)-2,5-diphenyltrazolium bromide(MTT) assay for instance (see Chang et al., PNAS 102, 18694-18699(2005)). MTT is a yellow tetrazolium salt reduced in metabolicallyactive cells to form insoluble purple formazan crystals, which aresolubilized by the addition of a detergent. The color can then bequantified by spectrophotometric means. These cells can be used todetermine whether ApoE(1-272)-specific anti-Apo E antibodies block thetoxicity of the fragment in this neuronal cell line. Antibody uptake ifneeded can occur by a mechanism such as micropinocytosis or pinocytosis.A read-through antibody (i.e., antibody with an epitope bridging thesite of truncation) can be used as a negative control. A positiveoutcome is increased MTT signal in antibody treated cells relative tountreated controls (negative control) or similar signal relative tountransfected cells (positive control) (i.e., preservation of metabolicactivity).

A similar assay can be performed with Neuro2A cells transfected withApoE(1-272). These cells produce the ApoE1-272 truncated fragmentdirectly without proteolytic process.

Example 3 In Vivo Testing of Antibodies

Antibodies preferentially binding to ApoE(1-272) over ApoE(1-299) oragents inducing such antibodies can be administered to human ApoE4containing mice on an APP transgenic background. Holtzman et al., PNAS2000 vol. 97 no. 6 2892-2897 described an example of such mice in whichmice having a knocked out endogenous Apo alleles are transfected withhuman APP V717F and human ApoE4. Immunohistological staining of thetransgenic mouse brain and western blots of these mice show increasedlevels of ApoE(1-272) fragment in the absence of such treatment andleads to behavioral deficits in the mice. Reduced levels of ApoE(1-272), increased synapotophysin levels, or inhibition of behavioraldeficits can therefore be used as efficacy endpoints.

Example 4 Immunoprecipitation Blotting

Solutions and Reagents:

The following solutions were prepared with Milli-Q or equivalentlypurified water: (1) 1× Phosphate Buffered Saline (PBS); (2) 1× CellLysis Buffer: 20 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1%Triton X-100, 2.5 mM Sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mMNa₃VO4, 1 μg/ml Leupeptin. 1 mM PMSF was added to the cell lysis bufferimmediately prior to use; and (3) 3×SDS Sample Buffer: 187.5 mM Tris-HCl(pH 6.8 at 25° C.), 6% w/v SDS, 30% glycerol, 150 mM DTT, 0.03% w/vbromophenol blue. Protein G Agarose Beads were used forimmunoprecipitation.

Cell Lysates Preparation:

Media was aspirated. Cells were treated for desired time by adding freshmedia containing regulator. Media was then removed and cells rinsd withice-cold PBS to harvest cells under nondenaturing conditions. PBS wasremoved, and 0.5 ml ice-cold 1× cell lysis buffer was added to eachplate (10 cm). The plates were incubated on ice for 5 minutes. Cellswere scraped off the plates and transferred to microcentrifuge tubes.The cells were kept on ice, and sonicated on ice three times for 5seconds each. The samples were then microcentrifuged for 10 minutes at14,000×g, 4° C., and the supernatant was transferred to a new tube. Whennecessary, lysate was stored at −80° C.

Immunoprecipitation and Blotting:

A lysate pre-clearing step was performed to reduce non-specific bindingto the Protein A/G agarose beads. Specifically, 20 μl agarose beadsslurry was added to 250 μl cell lysate. The mixture was incubated at 4°C. for 30 minutes, and spun at maximum speed for 2 minutes. Thesupernatant was recovered for antibody in next step.

About 250 μl cell lysate was mixed with 5 μg of primary antibody 15G8.The mixture was incubated with gentle rocking overnight at 4° C. ProteinG agarose beads (20 μl of 50% bead slurry) was then added and themixture was incubated with gentle rocking for another 1-3 hours at 4° C.The sample was microcentrifuged for 30 seconds at 4° C. Pellet waswashed five times with 500 μl of 1× cell lysis buffer. The sample waskept on ice during washes.

The pellet was resuspended with 20 μl 3×SDS sample buffer. Theresuspended pellet was vortexed, and then microcentrifuged for 30seconds. The sample was heated to 95-100° C. for 2-5 minutes andmicrocentrifuged for 1 minute at 14,000×g. About 15-30 μl sample wasloaded on SDS-PAGE gel (12-15%). The electrophoresis was carried out at150 Volts. The SDS-PAGE gel was electrotransferred to a nitrocellulosemembrane. The membrane was incubated in 25 ml of blocking buffer for 1hour at room temperature, and washed three times for 5 minutes each with15 ml of TBS/T.

The membrane was then incubated with primary antibody anti-ApoE4antibody 1F9 (MBL International Corp., Cat# M067-3) in 10 ml primaryantibody dilution buffer with gentle agitation overnight at 4° C. Themembrane was then washed three times for 5 minutes each with 15 ml ofTBS/T. Afterwards, the membrane was incubated with 10 ml Goat anti-MouseIgG_IRDye 800CW antibody (L1-cor Bio #926-32210) 1:20000 in dilutionbuffer with gentle agitation for 1 hour at room temperature, and washedthree times for 5 minutes each with 15 ml of TBS/T. The membrane wasscanned using L1-Cor Odyssey Imager.

Lysates of full-length ApoE3, full-length ApoE4, ApoE3(1-272), andApoE4(1-272) lysates were detected using Western blot with 1 μg/ml 1F9(Medical and Biological Laboratories) (Lanes 1-4; FIG. 3). In addition,lysates of full-length ApoE3, full-length ApoE4, ApoE3(1-272), andApoE4(1-272) were immununoprecipitated with m15G8 antibody. The m15G8immunoprecipitation were then detected using Western blot with 1 μg/ml1F9 (Lanes 5-8; FIG. 3). 1F9 is a ApoE4-specific antibody that does notbind ApoE3 proteins (FIG. 3; Lanes 1, 2, 5, and 6). The results wereshown in FIG. 3: Lane 1: Full-length ApoE3 lysate; Lane 2: ApoE3 1-272lysate; Lane 3: Full-length ApoE4 lysate; Lane 4: ApoE4 1-272 lysate;Lane 5: Full-length ApoE3 immunoprecipitated with mouse 15G8; Lane 6:ApoE3 1-272 immunoprecipitated with mouse 15G8; Lane 7: Full-lengthApoE4 immunoprecipitated with mouse 15G8; Lane 8: ApoE4 1-272immunoprecipitated with mouse 15G8.

The m15G8 immunoprecipitation of ApoE4 (1-272) was detected by 1F9 (Lane8; FIG. 3). However, blotting with 1F9 fails to detect any m15G8immunoprecipitation of full-length ApoE4 (Lane 7; FIG. 3). These resultsdemonstrate that 15G8 is specific for 1-272.

Example 5 Characterization of 12C12, 15E10, and 15G8 by Western Blot

Cortical tissue lysates from wild-type mouse, ApoE KO mouse, andtransgenic mice that express human ApoE isoform knock-in (E2, E3, and E4strains) and human Amyloid beta were analyzed using western blot.Monoclonal antibodies 12C12, 15E10, and 15G8 recognize multiple bands intissue lysates of different strains, including apoE KO mouse which serveas negative control/background reactivity of the mAbs (FIG. 4). Theresults were shown in FIG. 4: Lane 1: western blot of cortical lysatesfrom apoE KO mouse; Lane 2: recombinant full length ApoE4; Lane 3:recombinant full length ApoE3; Land 4: recombinant full length ApoE2;Lane 5: western blot of cortical lysates from wild-type mouse; Lane 6:western blot of cortical lysates from transgenic E4 knock-in mouse; Lane7: western blot of cortical lysates from transgenic E3 knock-in mouse;Lane 8: western blot of cortical lysates from transgenic E2 knock-inmouse.

In cortical lysates of wild-type, E3 or E4 knock-in mouse (but not incortical lysates of E2 or ApoE KO mouse), all three antibodies recognizea band around 30 KDa (Lanes 5, 6, and 7). In cortical lysates from E3 orE4 knock-in mouse (but not in tissue lysates of wild-type, E2 knock-in,or ApoE KO mouse), all three antibodies recognize a band around 60 KDa(Lanes 6 and 7). The molecular weights of these bands specific towild-type, E3 and E4 knock-in cortical lysates are consistent with thoseof ApoE(1-272) monomer (31.5 KDa) and dimer (63 KDa) (FIG. 4).

Example 6 Design of Humanized 15G8 Antibodies

The starting point or donor antibody for humanization is the mouseantibody 15G8. The variable kappa (Vκ) of m15G8 belongs to mouse Kabatsubgroup 2 which corresponds to human Kabat subgroup 2. The VH of m15G8belongs to mouse Kabat subgroup 3d which corresponds to human Kabatsubgroup 3 (Kabat et al., Sequences of Proteins of ImmunologicalInterest, Fifth Edition. NIH Publication No. 91-3242, 1991). Kabatnumbering is used throughout in this Example.

The 16-residue CDR-L1 belongs to canonical class 4, the 7-residue CDR-L2belongs to class 1, the 9-residue CDR-L3 belongs to class 1 in Vk(Martin & Thornton, J Mol. Biol. 263:800-15, 1996). The 5-residue CDR-H1belongs to class 1, the 16-residue CDR-H2 belongs to class 3 (Martin &Thornton, J Mol. Biol. 263:800-15, 1996). CDR-H3 has no canonicalclasses. The residues at the interface between the Vκ and VH domains arethe ones commonly found.

A search was made over the protein sequences in the PDB database(Deshpande et al., Nucleic Acids Res. 33: D233-7, 2005) to findstructures that would provide a rough structural model of 15G8. Thecrystal structure of dimeric antibody 4-B8(8)/E9 (pdb code 1KFA; Murataet al, Biochem. Biophys. Res. Commun. 293:489-496, 2002) was chosen forthe Vh structure since it has good overall sequence similarity andreasonable resolution (2.8

). In addition, CDRs-H1 and H2 of 1KFA have the same canonicalstructures as those of m15G8 Vh. 1KFA has an insertion in CDR-H3 at 100Dposition. The crystal structure of Nq16-113.8 (pdb code 2CJU_L; Scotti &Gherardi, J. Mol. Biol., 359:1161, 2006) was chosen for the Vk structuresince it had good overall sequence similarity and reasonably goodresolution (2.5

). Additionally, CDRs-L1, L2 and L3 had the same canonical structures asm15G8 Vk. A structural model of the mouse 15G8 Fv region was built usingthe 1KFA and 2CJU structures as templates in the PRIME modeling workflow(PSP 3.0111) within Schrodinger Suite2011 (update release, September2012). The structural model for CDR3 of the heavy chain was furtherrefined by Schrodinger loop search for the region comprising residues99-107. Ten iterations of extended loop sampling yielded oneenergetically preferred loop conformation in the context of the whole Fvregion.

A search of the non-redundant protein sequence database from NCBIallowed selection of suitable human frameworks into which to graft themurine CDRs. For Vk, a human kappa light chain with GenBank accessioncode AAT86035 (GI:50898163) was chosen. It belongs to Kabat human kappasubgroup 2 and has the same canonical classes for CDR-L1, L2 and L3 asthose of m15G8. For Vh, human Ig heavy chain AAX82494 (GI: 62421461)(Lundquist et al, Infect. Immun. 74 (6), 3222-3231, 2006) was chosen. Itbelongs to Kabat human heavy subgroup 3 and has the same canonicalclasses for CDR-H1 and H2 as those of m15G8. CDR-H3 of 15G8 has nocanonical class.

> AAX82494_VH_HuFwr (SEQ ID NO: 7)QVQLQESGGGLVKPGGSLKLSCAASGFTFSSYGMSWVRQTPDKRLEWVATISSGGSYTYYPDSVKGRFTISRDNAKNTLYLQMSSLKSEDTAMYYCARLYYGYRYYFDYWGQGTM VTVSS >AAT86035_VL_HuFwr (SEQ ID NO: 8)DVVMTQSPLSLPVTLGQPASISCRSSQSLLHSDGNTYLLWFLQRPGQSPRRLLYKVSDRDSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQGTHWPWTFGQGTKVEIK

The rationales for selection of several positions as candidates forbackmutation are as follows.

Variable Light Chain Backmutations:

V3L (here as elsewhere for framework backmutations, the first mentionedresidue is the human residue and the second the mouse residue): In thestructure model, Leucine in this position packs close to the CDR andthere is a high probability that this residue makes a contact withantigen. Therefore we make this back mutation.

F36Y: This is an interface residue. In the structure model, this residueis in close contact with heavy chain W103 interface residue. Thereforewe make this back mutation.

R46L: This is an interface residue. In the structure model, Leu packsbeneath CDRL2 and CDRH3, replacement with Arg will distort theconformation of these two CDRs.

Variable Heavy Chain Backmutations:

Q1E, Q3K & Q5V: In the structure model, Lysine at Kabat position H3 isin contact with CDRH1. In the structure, these three residues packtogether, i.e., both Glu at Kabat position H1 and Val at Kabat positionH5 interact with Lysine at Kabat position H3. Therefore these threeresidues are back mutated together.

D42E: Glu at Kabat position H42 is a more frequent residue in humanframeworks at this position than Aspartate, therefore it is backmutated.

K83R: Arg is more frequent in human frameworks at Kabat position H83,therefore it is back mutated.

Two humanized heavy chains and two humanized light chains were madeincorporating various back mutations (FIGS. 1-2 and Tables 1-2). Theamino acids at L3, L36, L46, H1, H3, H5, H42, and H83 in Hu15G8VLv1-v2and Hu15G8VHv1-v2 are listed in Tables 3-4.

TABLE 1 VH Backmutations VH exon donor VH variant acceptor sequenceframework residues Hu15G8VHv1 AAX82494 H1, H3, H5, H42, H83 SEQ ID NO:11 SEQ ID NO: 7 Hu15G8VHv2 AAX82494 none SEQ ID NO: 17 SEQ ID NO: 7

TABLE 2 VL Backmutations VL exon donor VL variant acceptor sequenceframework residues Hu15G8VLv1 AAT86035 L3, L36, L46, SEQ ID NO: 14 SEQID NO: 8 Hu15G8VLv2 AAT86035 none SEQ ID NO: 18 SEQ ID NO: 8

>15G8vh Version1 Amino acid sequence: (SEQ ID NO: 11)EVKLVESGGGLVKPGGSLKLSCAASGFTFS FYAMS WVRQTPEKRLEWVA SLSRGGSTY YPDSVKGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCAR EGATALYAMDY WGQGTM VTVSSNucleic acid sequence: (SEQ ID NO: 12)GAGGTGAAGCTGGTGGAGTCCGGCGGCGGCCTGGTGAAGCCCGGCGGCTCCCTGAAGCTGTCCTGCGCCGCCTCCGGCTTCACCTTCTCCTTCTACGCCATGTCCTGGGTGCGCCAGACCCCCGAGAAGCGCCTGGAGTGGGTGGCCTCCCTGTCCCGCGGCGGCTCCACCTACTACCCCGACTCCGTGAAGGGCCGCTTCACCATCTCCCGCGACAACGCCAAGAACACCCTGTACCTGCAGATGTCCTCCCTGCGCTCCGAGGACACCGCCATGTACTACTGCGCCCGCGAGGGCGCCACCGCCCTGTACGCCATGGACTACTGGGGCCAGGGCACCATGGTGACCGTGTCCTCC >15G8Vκ Version1 Amino acid sequence: (SEQ ID NO: 14)DVLMTQSPLSLPVTLGQPASISC RSSQSIVHSNGNTYLQ WYLQRPGQSPRLLLY KVSNR FSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC FQGSHVPWT FGQGTKVEIKNucleic acid sequence: (SEQ ID NO: 15)GACGTGCTGATGACCCAGTCCCCCCTGTCCCTGCCCGTGACCCTGGGCCAGCCCGCCTCCATCTCCTGCCGCTCCTCCCAGTCCATCGTGCACTCCAACGGCAACACCTACCTGCAGTGGTACCTGCAGCGCCCCGGCCAGTCCCCCCGCCTGCTGCTGTACAAGGTGTCCAACCGCTTCTCCGGCGTGCCCGACCGCTTCTCCGGCTCCGGCTCCGGCACCGACTTCACCCTGAAGATCTCCCGCGTGGAGGCCGAGGACGTGGGCGTGTACTACTGCTTCCAGGGCTCCCACGTGCCCTGGACCTTCGGCCAGGGCACCAAGGTGGAGATCAAG >15G8vh Version2 (No backmutation)(SEQ ID NO: 17) QVQLQESGGGLVKPGGSLKLSCAASGFTFS

WVRQTPDKRLEWVA

RFTISRDNAKNTLYLQMSSLKSEDTAMYYCAR

WGQGTMV TVSS >15G8Vκ Version2 (No backmutation) (SEQ ID NO: 18)DVVMTQSPLSLPVTLGQPASISC

WFLQRPGQSPRRLLY

GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYC

FGQGTKVEIK

TABLE 3 Kabat numbering of preferred framework residues for backmutationin humanized 15G8 antibody heavy chains mouse 15G8 Hu15G8VH2 AAX82494heavy chain Hu15G8VH1 SEQ ID SEQ ID NO: 7 SEQ ID NO: 9 SEQ ID NO: 11 NO:17 H1 Q E E Q H3 Q K K Q H5 Q V V Q H42 D E E D H83 K R R K

TABLE 4 Kabat numbering of preferred framework residues for backmutationin humanized 15G8 antibody light chains AAT86035 Mouse 15G8 Hu15G8VL2SEQ ID light chain Hu15G8VL1 SEQ ID NO: 8 SEQ ID NO: 10 SEQ ID NO: 14NO: 18 L3 V L L V L36 F Y Y F L46 R L L R

All publications (including GenBank Accession numbers,UniProtKB/Swiss-Prot accession numbers and the like), patents and patentapplications cited are herein incorporated by reference in theirentirety for all purposes to the same extent as if each individualpublication, patent and patent application was specifically andindividually indicated to be incorporated by reference in its entiretyfor all purposes. In the event of any variance in sequences associatedwith Genbank and UniProtKB/Swiss-Prot accession numbers and the like,the application refers to the sequences associated with the citedaccession numbers as of the filing date of the application.

1. A monoclonal antibody that preferentially binds to ApoE(1-272)relative to binding to ApoE(1-299).
 2. The monoclonal antibody of claim1 that binds to an epitope including residue 272 of ApoE(1-272).
 3. Themonoclonal antibody of claim 1 that binds to an epitope including a freecarboxyl group of position 272 of ApoE(1-272). 4-9. (canceled)
 10. Themonoclonal antibody of claim 1 having the three light chain CDRs asdefined by Kabat and three heavy chain CDRs as defined by Kabat ofmonoclonal antibody 12C12, 15E10 or 15G8.
 11. The antibody of claim 16,comprising a mature heavy chain variable region comprising the threeKabat CDRs of SEQ ID NO:9, and having at least 90% sequence identity toSEQ ID NO:9, and a mature light chain variable region comprising thethree Kabat CDRs of SEQ ID NO:10, and having at least 90% sequenceidentity to SEQ ID NO:10. 12-13. (canceled)
 14. The antibody of claim 1,wherein the mature heavy chain variable region comprises an amino acidsequence of SEQ ID NO:9 and the mature light chain variable regioncomprises an amino acid sequence of SEQ ID NO:10.
 15. The monoclonalantibody of any one of claims 1, 2 or 3 that is a humanized, chimeric,veneered or human antibody.
 16. The monoclonal antibody of claim 1, thatis an Fab fragment, single chain Fv, or single domain antibody. 17.(canceled)
 18. The monoclonal antibody of claim 1, wherein the isotypeis human IgG1. 19-21. (canceled)
 22. The antibody of claim 21 comprisinga light chain variable region comprising three light chain Kabat CDRs ofSEQ ID NO:18 and a heavy chain variable region comprising three heavychain CDRs of SEQ ID NO:17. 23-26. (canceled)
 27. The antibody of claim1, wherein the mature heavy chain variable region has at least 90%sequence identity to SEQ ID NO:17, and the mature light chain variableregion has at least 90% sequence identity to SEQ ID NO:18. 28-30.(canceled)
 31. The antibody of claim 27, wherein the mature heavy chainvariable region has at least 95% sequence identity to SEQ ID NO:17 andthe mature light chain variable region has at least 95% sequenceidentity to SEQ ID NO:18. 32-33. (canceled)
 34. The antibody of claim27, provided that position L3 (Kabat numbering) is occupied by L. 35.The antibody of claim 27, provided that position L36 (Kabat numbering)is occupied by Y.
 36. The antibody of claim 27, provided that positionL46 (Kabat numbering) is occupied by L.
 37. The antibody of claim 27,provided that position H1 (Kabat numbering) is occupied by E.
 38. Theantibody of claim 27, provided that position H3 (Kabat numbering) isoccupied by K.
 39. The antibody of claim 27, provided that position H5(Kabat numbering) is occupied by V.
 40. The antibody of claim 27,provided that position H42 (Kabat numbering) is occupied by E.
 41. Theantibody of claim 27, provided that position H83 (Kabat numbering) isoccupied by R.
 42. The antibody of claim 27, wherein the amino acidsequence of the mature heavy chain variable region is SEQ ID NO:17 andthe amino acid sequence of the mature light chain variable region is SEQID NO:18 provided that position L3 (Kabat numbering) can be occupied byV or L, position L36 (Kabat numbering) can be occupied by F or Y,position L46 (Kabat numbering) can be occupied by R or L, position H1(Kabat numbering) can be occupied by Q or E, position H3 (Kabatnumbering) can be occupied by Q or K, position H5 (Kabat numbering) canbe occupied by Q or V, position H42 (Kabat numbering) can be occupied byD or E, and position H83 (Kabat numbering) can be occupied by K or R.43. The antibody of claim 42, wherein position L3 (Kabat numbering) isoccupied by L, position L36 (Kabat numbering) is occupied by Y, andposition L46 (Kabat numbering) is occupied by L.
 44. The antibody ofclaim 42, wherein position L3 (Kabat numbering) is occupied by V,position L36 (Kabat numbering) is occupied by F, and position L46 (Kabatnumbering) is occupied by R.
 45. The antibody of claim 42, whereinposition H1 (Kabat numbering) is occupied by E, position H3 (Kabatnumbering) is occupied by K, position H5 (Kabat numbering) is occupiedby V, position H42 (Kabat numbering) is occupied by E, and position H83(Kabat numbering) is occupied by R.
 46. The antibody of claim 42,wherein position H1 (Kabat numbering) is occupied by Q, position H3(Kabat numbering) is occupied by Q, position H5 (Kabat numbering) isoccupied by Q, position H42 (Kabat numbering) is occupied by D, andposition H83 (Kabat numbering) is occupied by K. 47-54. (canceled) 55.The antibody of claim 27, wherein the mature heavy chain variable regionhas an amino acid sequence of SEQ ID NO:11 and the mature light chainvariable region has an amino acid sequence of SEQ ID NO:14.
 56. Theantibody of claim 27, wherein the mature heavy chain variable region hasan amino acid sequence of SEQ ID NO:17 and the mature light chainvariable region has an amino acid sequence of SEQ ID NO:18.
 57. Theantibody of claim 27, wherein the mature heavy chain variable region hasan amino acid sequence of SEQ ID NO:11 and the mature light chainvariable region has an amino acid sequence of SEQ ID NO:18.
 58. Theantibody of claim 27, wherein the mature heavy chain variable region hasan amino acid sequence of SEQ ID NO:17 and the mature light chainvariable region has an amino acid sequence of SEQ ID NO:14.
 59. Theantibody of claim 27, provided that any differences in CDRs of themature heavy chain variable region and mature light variable region fromSEQ ID NO:17 and SEQ ID NO:18, respectively reside in positions1160-1165 (Kabat numbering).
 60. The antibody of claim 27, wherein themature heavy chain variable region is fused to a heavy chain constantregion and the mature light chain constant region is fused to a lightchain constant region.
 61. (canceled)
 62. The antibody of claim 27,wherein the heavy chain constant region is of human IgG1m3 allotype. 63.The antibody of claim 60, wherein the heavy chain constant region hasthe amino acid sequence of SEQ ID NO:39 provided the C-terminal lysineresidue may be omitted.
 64. The antibody of claim 63, wherein the heavychain constant region has the amino acid sequence of SEQ ID NO:39. 65.The antibody of claim 60, wherein the heavy chain constant region hasthe amino acid sequence of SEQ ID NO:38 provided the C-terminal lysineresidue may be omitted.
 66. The antibody of claim 60, wherein the heavychain constant region has the amino acid sequence of SEQ ID NO:37provided the C-terminal lysine residue may be omitted.
 67. The antibodyof claim 60, wherein the heavy chain constant region has the amino acidsequence of SEQ ID NO:40 provided the C-terminal lysine residue may beomitted.
 68. The antibody of claim 60, wherein the light chain constantregion has the amino acid sequence of SEQ ID NO:35.
 69. The antibody ofclaim 55, wherein the mature heavy chain variable region is fused to aheavy chain constant region having the amino acid sequence of SEQ IDNO:39 provided the C-terminal lysine residue may be omitted and themature light chain constant region is fused to a light chain constantregion having the amino acid sequence of SEQ ID NO:35.
 70. The antibodyof claim 27, wherein the mature light chain has the amino acid sequenceof SEQ ID NO:16 and a the mature heavy chain has the amino acid sequenceof SEQ ID NO:13. 71-72. (canceled)
 73. A nucleic acid encoding a matureheavy chain variable region and/or a mature light chain variable regionas defined by any of claims 27, 42, 55, 56, 57 or
 58. 74. The nucleicacid of claim 73 having a sequence comprising any one of SEQ ID NO:12and SEQ ID NO:15.
 75. (canceled)
 76. A method of humanizing an antibody,comprising determining the sequences of the heavy and light chainvariable regions of a mouse antibody, synthesizing a nucleic acidencoding a humanized heavy chain comprising CDRs of the mouse heavychain and a nucleic acid encoding a humanized light chain comprisingCDRs of the mouse light chain; expressing the nucleic acids in a hostcell to produce a humanized antibody, wherein the mouse antibody is12C12, 15E10 or 15G8, characterized by a light chain variable region ofSEQ ID NO:10 and a heavy chain variable region of SEQ ID NO:9.
 77. Amethod of producing a humanized, chimeric or veneered antibody,comprising culturing cells transformed with nucleic acids encoding theheavy and light chains of the antibody, so that the cells secrete theantibody; and purifying the antibody from cell culture media; whereinthe antibody is a humanized, chimeric or veneered form of 12C12, 15E10or 15G8, wherein 12C12, 15E10 or 15G8 is a mouse antibody characterizedby a light chain variable region of SEQ ID NO:10 and a heavy chainvariable region of SEQ ID NO:9.
 78. A method of producing a cell lineproducing a humanized, chimeric or veneered antibody, comprising:introducing a vector encoding heavy and light chains of an antibody anda selectable marker into cells; propagating the cells under conditionsto select for cells having increased copy number of the vector;isolating single cells from the selected cell; and banking cells clonedfrom a single cell selected based on yield of antibody; wherein theantibody is a humanized, chimeric or veneered form of 12C12, 15E10 or15G8, wherein 12C12, 15E10 or 15G8 is a mouse antibody characterized bya light chain variable region of SEQ ID NO:10 and a heavy chain variableregion of SEQ ID NO:9. 79-81. (canceled)
 82. A pharmaceuticalcomposition comprising the antibody of claim 1 and a pharmaceuticallyacceptable carrier.
 83. An isolated fragment of ApoE including 3-10contiguous residues of ApoE and having a C-terminus ending at residue272.
 84. The isolated fragment of claim 83, linked to a carrier moleculeoptionally via a spacer that helps elicit antibodies against thefragment.
 85. The isolated fragment of claim 84, consisting essentiallySEQ ID NO:41.
 86. A pharmaceutical composition comprising the fragmentof claim 84 and an adjuvant acceptable for administration to humans.87-91. (canceled)
 92. A method of treating or effecting prophylaxis of adisease associated with ApoE4 comprising administering an effectiveregime of an antibody that preferentially binds to ApoE(1-272) relativeto ApoE(1-299) or an agent that induces such an antibody to a patienthaving or at risk of the disease and thereby treating or effectingprophylaxis of the disease. 93-103. (canceled)
 104. A method ofscreening an agent for activity against Alzheimer's disease, comprisingadministering the agent to a transgenic animal expressing ApoE and APPtransgenes, and determining whether the agent inhibits or delays atleast one sign or symptom of Alzheimer's disease, wherein the agent isan antibody that preferentially binds to ApoE(1-272) relative to ApoE(1-299) or an agent that induces such an antibody.
 105. A method ofgenerating an antibody that preferentially binds to ApoE(1-272) relativeto binding to ApoE(1-299), comprising immunizing an animal with animmunogen comprising a fragment of ApoE consisting of SEQ ID NO:41linked to a carrier on the N-terminus of the fragment.
 106. The methodof claim 105, wherein the immunogen comprises SEQ ID NO:5. 107-109.(canceled)